Regulation of plasma retinol binding protein secretion in human HepG2 cells

Retinol binding protein (RBP) is the plasma transport protein of retinol. Mobilization of RBP from the liver stores is stimulated by retinol. During vitamin A deficiency, RBP secretion is specifically inhibited while its rate of biosynthesis is unaffected. As a consequence, RBP, as apoprotein, accumulates inside the endoplasmic reticulum (ER) of the hepatocyte, and a new elevated steady-state concentration is reached. We have studied the role of degradation on the regulation of RBP metabolism in retinol deficient HepG2 cells and determined the intracellular site where RBP degradation takes place. Pulse-chase experiments show that RBP half-life is ca.9 h in retinol-depleted cells. RBP degradation is slow and is insensitive to the treatment with NH4Cl, which inactivates lysosomal proteases and to the drug brefeldin A, which prevents protein export from the ER. The data obtained suggest that RBP degradation occurs, at least in part, in a pre-Golgi compartment. 2-Mercaptoethanol, at millimolar concentration, induces RBP secretion, suggesting a possible role for sulfhydryl-mediated apo-RBP retention by resident ER proteins.     

Trafficking of exogenous fatty acids within Caco-2 cells.

Dietary fatty acids (FAs) crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane. This apparent compartmentalization of FA metabolism in enterocytes was further investigated using a model human enterocyte-like intestinal cell line. [3H]Oleic acid bound to bovine serum albumin (BSA) was added to the apical or basolateral surfaces of confluent monolayers of Caco-2 cells growing on uncoated polycarbonate filters. In other experiments, [3H]oleic acid incorporated into micelles with taurocholate (+/- 2-monoacylglycerol) was added apically. Caco-2 cells absorbed oleic acid bound to BSA from both the apical and basolateral surfaces at the same rate. Oleic acid in micellar solution was absorbed more efficiently than oleic acid bound to BSA. Regardless of its site or mode of presentation, the majority of the incorporated oleic acid was found in triglycerides. Only a small fraction was subjected to beta-oxidation or esterification into phospholipids. Most of the incorporated oleic acid was still retained intracellularly at 24 h. The polarity of triglyceride secretion was influenced by the experimental conditions. Triglyceride secretion was not significantly polarized when oleic acid-BSA was presented apically. However, the ratio of basolateral to apical secretion at 24 h was 9:1 for oleic acid-BSA presented basolaterally. For oleic acid in taurocholate micelles there was a trend toward polarity of secretion to the apical media (apical to basolateral ratio = 2:1). The inclusion of 2-monoacylglycerol in oleic acid-taurocholate micelles did not augment triglyceride synthesis or secretion. These differences indicate that compartmentation of FA metabolism in Caco-2 cells is influenced by the site of FA presentation. Northern and Western blot hybridization studies indicated that the liver fatty acid-binding protein but not the intestinal fatty acid-binding protein gene is expressed in these cells. The absence of this latter 15 kDa protein indicates that it is not required by Caco-2 cells for the synthesis of triglycerides or for the polarized export of triglyceride. These studies indicate that the Caco-2 cell line will be a useful model system for studying the polarization of FA trafficking/metabolism in enterocytes and defining the role of intracellular fatty acid binding proteins in these processes.     

Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification.

The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids. These differences stem from the unique physical and chemical properties of long-chain fatty acids. To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane. More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered. Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics. Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics. In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase [AMP forming] [EC 6.2.1.3]). FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional. This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids.     

The effects of unsaturated fatty acids on lipid metabolism in HepG2 cells

We investigated the effects of stearic acid (saturated), oleic acid (monounsaturated), linoleic acid (n-6 polyunsaturated), and alpha-linolenic acid (n-3 polyunsaturated) on lipid metabolism in a hepatocyte-derived cell line, HepG2. HepG2 cells were cultured in medium supplemented with either stearic acid (0.1% w/v), oleic acid (0.1% v/v), linoleic acid (0.1% v/v), or alpha-linolenic acid (0.1% v/v). After 24 h, expression of lipid metabolism-associated genes was evaluated by real-time PCR. Alpha-linolenic acid showed a suppressive effect on the hepatic fatty acid de novo synthesis and fatty acid oxidation pathways, while linoleic acid also showed a tendency to suppress these pathways although the effect was weaker. Moreover, alpha-linolenic acid enhanced the expression of enzymes associated with reactive oxygen species (ROS) elimination. In contrast, oleic acid tended to promote fatty acid synthesis and oxidation. In conclusion, alpha-linolenic acid and linoleic acid may be expected to ameliorate hepatic steatosis by downregulating fatty acid de novo synthesis and fatty acid oxidation, and by upregulating ROS elimination enzymes. Oleic acid had no distinct effects for improving steatosis or oxidative stress.     

Suppression of ABCA1 by unsaturated fatty acids leads to lipid accumulation in HepG2 cells

Abnormal lipid metabolism may contribute to the pathogenesis of non-alcoholic steatohepatitis (NASH). ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins. We previously reported that unsaturated fatty acids destabilise ABCA1 in murine macrophages and ABCA1-transfected baby hamster kidney cells by increasing its protein degradation. Here, we examined the correlation between ABCA1 and hepatic lipids. In HepG2 cells, unsaturated but not saturated fatty acids suppressed ABCA1 protein levels by promoting its protein degradation. Over-expression of ABCA1 resulted in a decrease of cellular fatty acids and triglycerides, while repression by ABCA1 siRNA increased both cellular fatty acids and triglycerides. Rats with NASH also showed lower ABCA1 protein levels in liver cells, compared with that of the normal rats. These data indicate that steatosis is associated with a decrease in ABCA1 protein expression leading to an increase in lipid storage in hepatocytes. And it further suggests that this effect could be due to an excess of unsaturated fatty acids.     

Enzymatic esterification of exogenous retinol and 3,4-didehydroretinol in the retinal pigment epithelium

The kinetics of esterification of exogenous retinol by cell membranes prepared from the crude homogenate of the frog retinal pigment epithelium was studied. The formation of retinyl palmitate from added retinol was directly assayed by high performance liquid chromatography (HPLC). A linear relationship was observed between the amount of protein (up to 2 mg) in the incubation medium and the amount of retinyl palmitate formed. At room temperature, this reaction took less than 2 hours to complete. By varying the substrate concentration in the incubation medium, the reciprocal of initial velocity of the reaction (nmol retinyl palmitate formed per hour) was plotted against the reciprocal of substrate concentration (nmol of retinol). This double-reciprocal plot shows that the apparent Km of the reaction was 10 microM with an apparent Vmax of 9.1 nmol of retinyl palmitate per hour per mg protein. When this assay was repeated in the presence of 3,4-didehydroretinol (20 microM), the kinetics of the reaction showed the pattern of that of a competitive inhibitor, suggesting that 3,4-didehydroretinol competes with retinol for the same active site for esterification. The esterification of 3,4-didehydroretinol resulted in the formation of 3,4-didehydroretinyl palmitate, which was also measured by HPLC. The amount of 3,4-didehydroretinyl palmitate formed by this reaction decreased in proportion to increased retinol concentration in the incubation mixture. This further confirms that a competition exists between the esterification of retinol and 3,4-didehydroretinol by retinal pigment epithelium of the frog.     

Induction of retinol esterification in retinal pigment epithelial cells by butyrate

Butyrate induced marked morphological changes in cultured human retinal pigment epithelial (RPE) cells. The cells assumed a flattened structure within six hours of exposure to butyrate. The butyrate-treated retinal pigment epithelial cells possessed an enhanced capacity to esterify retinol. Among all short chain organic acids tested, butyrate was by far the most effective, followed by pentanoate and hexanoate. The inductive effect of butyrate was specific for retinol esterification, since the incorporations of fatty acid into phosphatidyl choline and cholesteryl ester were not enhanced. Time-dependent, butyrate-enhanced retinol esterification may be related to the inhibition of cell proliferation. This represents the first report on the induction of retinol esterification in cultured retinal pigment epithelial cells.     

Regulation of sulfate uptake by amino acids in cultured tobacco cells

The sulfur requirements of tobacco (Nicotiana tabacum L. var. Xanthi) XD cells grown in chemically defined liquid media can be satisfied by sulfate, thiosulfate, l-cyst(e)ine, l-methionine or glutathione, and somewhat less effectively by d-cyst (e) ine, d-methionine or dl-homocyst (e)ine. Sulfate uptake is inhibited after a 2 hr lag by l-cyst (e)ine, l-methionine, l-homocyst(e)ine or l-isoleucine, but not by any of the other protein amino acids, nor by d-cyst(e)ine. l-cyst(e)ine is neither a competitive nor a non-competitive inhibitor of sulfate uptake. Its action most closely resembles apparent uncompetitive inhibition. Inhibition of sulfate uptake by l-cyst(e)ine can be partially prevented by equimolar l-arginine, l-lysine, l-leucine, l-phenylalanine, l-tyrosine or l-tryptophan, but is little affected by any of the other protein amino acids. The effective amino acids are apparent competitive inhibitors of l-cyst(e)ine uptake after a 2 hr lag. Inhibition of sulfate uptake by l-methionine cannot be prevented, nor can uptake of l-methionine be inhibited by any single protein amino acid. The results suggest the occurrence of negative feedback control of sulfate assimilation by the end products, the sulfur amino acids, in cultured tobacco cells.     

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.     

Regulation of cholesterol esterification by micellar cholesterol in CaCo-2 cells

The regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol was studied in an established enterocyte cell line. CaCo-2 cells were grown in culture to confluency and dome formation. They were characterized morphologically by light and transmission electron microscopy. During the culture period, ACAT activity remained stable while the activities of the brush border enzymes sucrase and alkaline phosphatase progressively increased with time and plateaued 12 days after plating. As determined by the rate of incorporation of oleic acid into the individual lipid classes, the rate of triglyceride synthesis was twice that of phospholipid and 15 times that of cholesteryl ester synthesis in these cells. Incubating CaCo-2 cells with cholesterol solubilized in taurocholate micelles resulted in a significant increase in ACAT activity (149 +/- 5 pmol/dish per 2 hr vs. 366 +/- 5, (P less than 0.001) without changing the rates of triglyceride or phospholipid synthesis. The stimulation of ACAT activity by micellar cholesterol was rapid, occurring within 5 min and reaching a maximal effect by 2 hr. The regulation of ACAT activity by cholesterol was directly dependent upon the concentration of cholesterol solubilized in the micelle and was independent of protein synthesis. Incubating CaCo-2 cells with micellar cholesterol did not increase the esterification of, nor did the cholesterol enter the pool of, newly synthesized or performed cholesterol within 2 hr. The micellar cholesterol that was taken up by the cells was esterified within 5 min after starting the incubation. Progesterone, a known ACAT inhibitor, significantly decreased the rate of esterification of intracellular micellar cholesterol proving that the cholesterol taken up by CaCo-2 cells was indeed entering the ACAT pool. Despite increasing amounts of unesterified cholesterol entering the cells via micelles, the percent of cholesterol that was esterified at any one time remained constant at 1%. The results suggest that ACAT activity in CaCo-2 cells is stimulated by cholesterol delivered to the cells by way of taurocholate micelles. The rapid entry of this sterol into the ACAT substrate pool suggests that ACAT activity in CaCo-2 cells is regulated by the expansion of the cholesterol substrate pool that is being utilized by an unsaturated ACAT enzyme.     

Effect of formaldehyde and resveratrol on the viability of Vero, HepG2 and MCF-7 cells

A non-transformed (Vero) and two tumor cell lines (HepG2 and MCF-7) were treated with 10nM to 100 microM formaldehyde. Lower doses (10nM to 10 microM) enhanced the viability of the cultured cells, measured by MTT assay. Higher doses (75-100 microM) decreased viability of the cells by 50% or more. The 100 microM concentration of HCHO has been chosen for combination treatment of the three cell lines with a series of concentrations (0.2-100 microM) of resveratrol, a phytoestrogen occurring in various fruits. Resveratrol decreased the cytotoxicity of formaldehyde depending on cell line and point of time, especially in case of MCF-7 cells at 24 and 72 h, Vero cells at 24h and HepG2 cells at 48 h after treatment. Possible modes of interactions are discussed, considering the role of resveratrol in formaldehyde metabolism and also the estrogen receptor positivity of MCF-7 cells.     

Esterification of exogenous and endogenous fatty acids by rat adipocytes in vitro.

Rat adipocytes were used in vivo to compare the esterification of exogenous fatty acids and fatty acids formed de novo from glucose or acetate. Pure single fatty acids added to the medium were esterified at comparable rates but marked differences were observed when the same acids were supplied as components of a fatty acid mixture of a composition similar to that in the tissue. Fatty acids synthesised de novo from acetate by adipocytes in a medium containing high concentrations of acetate were located predominantly in diacylglycerols. The effect was most marked with adipocytes from older rats and was enhanced by the presence of exogenous long-chain fatty acids. Exogenous oleic acid was esterified predominantly into triacylglycerols at all concentrations of acetate. No such accumulation of endogenously-synthesised fatty acids in diacylglycerols occurred when glucose was the precursor for fatty acid synthesis. The diacylglycerols formed were almost entirely of the sn-1,2-configuration.     

Regulation of squalene epoxidase in HepG2 cells

Regulation of squalene epoxidase in the cholesterol biosynthetic pathway was studied in a human hepatoma cell line, HepG2 cells. Since the squalene epoxidase activity in cell homogenates was found to be stimulated by the addition of Triton X-100, enzyme activity was determined in the presence of this detergent. Incubation of HepG2 cells for 18 h with L-654,969, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, increased squalene epoxidase activity dose-dependently. On the other hand, low density lipoprotein (LDL) and 25-hydroxy-cholesterol decreased the enzyme activity. These results demonstrate that squalene epoxidase is regulated by the concentrations of endogenous and exogenous sterols. The affinity of the enzyme for squalene was not changed by treatment with L-654,969. Cytosolic (S105) fractions, prepared from HepG2 cells treated with or without L-654,969, had no effect on microsomal squalene epoxidase activity of HepG2 cells, in contrast to the stimulating effect of S105 fractions from rat liver homogenate. Mevalonate, LDL, and oxysterol treatment abolished the effect of L-654,969. Simultaneous addition of cycloheximide and actinomycin D also prevented enzyme induction in HepG2 cells. From these results, the change in squalene epoxidase activity is thought to be caused by the change in the amount of enzyme protein. It is further suggested that squalene epoxidase activity is suppressed only by sterols, not by nonsterol derivative(s) of mevalonate, in contrast to the regulation of HMG-CoA reductase.     

Regulation of cytoskeletal structure in human mammary carcinoma cells MCF-7 by culture substrata

Three-dimensional cellular structures formed by MCF-7 human mammary carcinoma cells within collagen gels were isolated with collagenase and cultivated on plastic substratum to examine whether the cytoskeleton specific for cells forming cellular structures (S-type) changes to that specific for cells grown as monolayers (M-type). The cytoskeleton isolated as 0.05% Triton-insoluble fraction from the cellular structures after culture for 1 day on plastic was exclusively S-type. However, both types of cytoskeletons were observed in the cellular structures cultivated for 7 days on plastic as well as in the cells grown as monolayers for 2 days after dissociation of the cellular structures with trypsin. By use of an antibody raised against a 65-kD polypeptide that was specific for the M-type cytoskeleton, the presence of the polypeptide was found to be restricted to the cells grown out as monolayers from the edge of the cellular structures. In the cells grown for 2 days as monolayers, a mixture of cells both having and lacking the polypeptide was observed. After a 7-day culture of the dissociated cells as monolayers on plastic, however, most of the cells had M-type cytoskeletons. The present results show that the apparent change in the cytoskeleton of MCF-7 cells from S-type to M-type does not occur in cells involved in the three-dimensional cellular structures even in the absence of collagen gels, but that it occurs in cells which are grown as monolayers for at least 7 days on plastic substratum.