Iron-ascorbate alters the efficiency of Caco-2 cells to assemble and secrete lipoproteins

F. Courtois1,2, I. Suc1, C. Garofalo1,2, M. Ledoux2, E. Seidman1,3, and E. Levy1,2

1 Centre de Recherche, Hôpital Sainte-Justine, and Departments of 2 Nutrition and 3 Pediatrics, University of Montreal, Montreal, Quebec, Canada H3T 1C5


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although oxidative stress has been implicated in development of gut pathologies, its role in intestinal fat transport has not been investigated. We assessed the effect of Fe2+-ascorbate-mediated lipid peroxidation on lipid synthesis, apolipoprotein biogenesis, and lipoprotein assembly and secretion. Incubation of postconfluent Caco-2 cells with iron(II)-ascorbate (0.2 mM/2 mM) in the apical compartment significantly promoted malondialdehyde formation without affecting sucrase activity, transepithelial resistance, DNA and protein content, and cell viability. However, addition of the oxygen radical-generating system reduced 1) [14C]oleic acid incorporation into cellular triglycerides (15%, P < 0.0002) and phospholipids (16%, P < 0.0005); 2) de novo synthesis of cellular apolipoprotein A-I (apo A-I) (18%, P < 0.05), apo A-IV (38%, P < 0.05), and apo B-48 (45%, P < 0.003) after [35S]methionine addition; and 3) production of chylomicrons (50%), VLDL (40%), LDL (37%), and HDL (30%) (all P < 0.0001). In contrast, increased total cellular cholesterol formation (96%, P < 0.0001), assayed by [14C]acetate incorporation, was noted, attributable to marked elevation (70%, P < 0.04) in activity of DL-3-hydroxy-3-methyl-glutaryl-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. The ratio of Acyl-CoA to cholesterol acyltransferase, the esterifying cholesterol enzyme, remained unchanged. Fe2+-ascorbate-mediated lipid peroxidation modifies intracellular fat absorption and may decrease enterocyte efficiency in assembling and transporting lipids during gut inflammation.

oxidative stress; intestine; apolipoproteins; fat malabsorption


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

REACTIVE OXYGEN SPECIES play an important role in health and disease (for review, see Refs. 17 and 20). They are generated as by-products of aerobic metabolism or produced by phagocytic cells (neutrophils, macrophages) as a nonspecific defense mechanism against intruding organisms (2, 7). The presence of potent cellular detoxification systems minimizes radical generation, terminates radical processes, and repairs damaged macromolecules (12). However, continued overproduction of reactive oxygen species and free radicals can overwhelm antioxidant defense and become deleterious to cellular biological processes and tissue functions. In this case, reactive oxygen species may cause transient or permanent damage to cellular constituents, including nucleic acids, proteins, lipids, and membranes (1, 39, 43). In addition, iron-catalyzed lipid peroxidation produces marked perturbations in plasma lipid transport and hepatobiliary sterol metabolism (6). Nevertheless, the mechanisms involved in these metabolic derangements remain obscure and warrant further studies using cell cultures to identify the specific organ(s) behind the reported qualitative and quantitative changes.

The gastrointestinal mucosa is constantly exposed to luminal oxidants from ingested foods (8, 13, 35). Foods of animal origin contain varying concentrations of lipid oxidation products, depending on the severity of processing and storage. For example, 30 types of cholesterol oxides have been found in cholesterol-containing foods for human consumption (34, 37, 44). Similarly, iron salts and ascorbic acid, frequently consumed together in multiple-vitamin preparations or ingested foods, form reactive hydroxyl radicals. Intraluminal catalase-negative bacteria produce large quantities of H2O2. Oxidase, such as xanthine oxidase from desquamated cells, also amplifies the generation of reactive oxygen metabolites (35, 36). Even saliva contains hypothiocyanous acid, formed from the interaction between salivary peroxidases with H2O2 and thiocyanate, which may increase luminal reactive oxygen metabolite content (35, 36). Clearly, the ingestion and/or occurrence of peroxides may have implications for human health, particularly in the long term. In this regard, reactive oxygen metabolites have been said to be responsible for injury to the intestinal mucosa in several disease states, including intestinal ischemia and subsequent reperfusion, as well as inflammatory bowel disease (30). As to the exact fate of fat uptake and transport after peroxidative attack directed toward the brush-border membrane, very little information is available. Although intestinal malabsorption has been reported in inflammatory bowel disease, no thorough investigation has been carried out to examine enterocyte lipid processing subsequent to lipid peroxidation. The present experiments were, therefore, undertaken to examine the possible effects of the Fe2+-ascorbate oxygen-radical generating system on lipid esterification and synthesis, apolipoprotein biogenesis, and lipoprotein assembly and secretion.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Caco-2 cells (American Type Culture Collection, Rockville, MD) were grown at 37°C with 5% CO2 in MEM (GIBCO BRL, Grand Island, NY) containing 1% penicillin/streptomycin and 1% MEM nonessential amino acids (GIBCO BRL) and supplemented with 10% decomplemented fetal bovine serum (FBS; Flow, McLean, VA). Caco-2 cells (passages 30-40) were maintained in T-75 cm2 flasks (Corning Glass Works, Corning, NY). Cultures were split (1:6) when they reached 70-90% confluence, using 0.05% trypsin-0.5 mM EDTA (GIBCO BRL). For individual experiments, cells were plated at a density of 1 × 106 cells/well on 24.5-mm polycarbonate Transwell filter inserts with 0.4-µm pores (Costar, Cambridge, MA), in MEM (as described above) supplemented with 5% FBS. The inserts were placed into six-well culture plates, permitting separate access to the upper and lower compartments of the monolayers. Cells were cultured for 20 days, a period at which the Caco-2 cells are highly differentiated and appropriate for lipid synthesis (31, 32). The medium was refreshed every second day.

Iron(II)-ascorbate (1:10, Sigma, Montreal, PQ, Canada) was then added to cells at different concentrations for 24 h. Sucrase activity was measured as a marker of cell differentiation and transepithelial resistance as a marker of monolayer integrity (Millipore, Bedford, MA) (31, 32). Cell viability was assessed by trypan blue exclusion (32). Furthermore, DNA and protein content was evaluated as previously described (29, 32). All these parameters were tested in the presence or absence of Fe2+-ascorbate.

Estimation of lipid peroxidation. Caco-2 cells were cultured in the presence or absence of Fe2+-ascorbate. The reaction was terminated by the addition of 0.2% butylated hydroxytoluene (2,6-di-t-butyl-p-cresol, BHT, Sigma) to measure malondialdehyde (MDA), as an index of lipid peroxidation. The amount of free MDA formed during the reaction was determined by HPLC as described previously (6). Proteins were first precipitated with a 10% sodium tungstate (Na2WO4) (Aldrich Chemical) solution, and protein-free supernatant was then reacted with an isovolume of 0.5% thiobarbituric acid (Sigma) solution at 90°C for 30 min. After cooling to room temperature, chromogene was extracted with 1-butanol and dried over a stream of nitrogen at 37°C. The dry extract was then resuspended in KH2PO4/methanol (70:30, pH 7.0) mobile phase before MDA detection by HPLC.

Measurement of lipid synthesis and secretion. Lipid synthesis and secretion were assayed as previously described (32, 42). Briefly, radiolabeled [14C]oleic acid (sp act, 53 mCi/mmol; Amersham, Oakville, ON, Canada) was added to unlabeled oleic acid and then solubilized in fatty acid-free BSA [BSA/oleic acid, 1:5 (mol:mol)]. The final oleic acid concentration was 0.7 mM (0.45 µCi)/well. Cells were first washed with PBS (GIBCO), and the [14C]oleic acid-containing medium was added to the upper compartment. Fe2+-ascorbate (0.2 mM:2 mM) was added to the upper chamber in serum-free MEM. At the end of a 24-h incubation period, cells were washed, then scraped with a rubber policeman in a PBS solution containing antiproteases (phenylmethylsulfonyl fluoride, pepstatin, EDTA, aminocaproic acid, chloramphenicol, leupeptin, glutathione, benzamidine, dithiothreitol, sodium azide, and Trasylol, all at a final concentration of 1 mM). An aliquot was taken for lipid extraction by standard methods (27) in the presence of unlabeled carrier [phospholipids (PL), monoglycerides, diglycerides, triglycerides (TG), free fatty acids, free cholesterol (FC), and cholesteryl ester (CE)].

The various lipid classes synthesized from [14C]oleic acid were then separated by TLC using the solvent mixture of hexane, ether, and acetic acid (80:20:3, vol/vol/vol), as previously described (27, 28). The area corresponding to each lipid was scratched off the TLC plates, and the silica powder was placed in a scintillation vial with Ready Safe counting fluid (Beckman, Fullerton, CA). Radioactivity was then measured by scintillation counting (LS 5000 TD, Beckman). Cell protein was quantified by the Bradford method, and results were expressed as dpm per milligram of cell protein. Lipid secreted in the basolateral compartment was analyzed and quantified, as described above, after centrifugation (2,000 rpm for 30 min at 4°C) to remove cell debris.

Cholesterol biogenesis was evaluated employing [14C]acetate as precursor (53.9 Ci/mmol) after a 24-h incubation period. Separation of FC and CE was performed by TLC.

Lipid carrier. Blood was drawn 2 h after the oral intake of a fat meal by human volunteers, and postprandial plasma was prepared to serve as a carrier for the lipoproteins synthesized by Caco-2 cells. The TG-enriched plasma was incubated at 56°C for 1 h to inactivate enzymatic activity in the presence of antiproteases.

Isolation of lipoproteins. For the determination of secreted lipoproteins, Caco-2 cells were incubated with the lipid substrate as described above, in the presence or absence of Fe2+-ascorbate. The medium supplemented with antiproteases (as described above) was first mixed with a plasma lipid carrier (4:1, vol/vol) to efficiently isolate de novo lipoproteins synthesized. The lipoproteins were then isolated by sequential ultracentrifugation using a TL-100 ultracentrigfuge (Beckman), as described previously (27, 28). Briefly, chylomicrons were isolated after ultracentrifugation (20,000 rpm for 20 min). Very low-density lipoprotein (VLDL) (1.006 g/ml) and low-density lipoprotein (LDL) (1.063 g/ml) were separated by spinning at 100,000 g for 2.26 h with a tabletop ultracentrifuge 100.4 rotor at 4°C. The high-density lipoprotein (HDL) fraction was obtained by adjusting the LDL infranatant to density at 1.21 g/ml and centrifuging for 6.5 h at 100,000 g. Each lipoprotein fraction was exhaustively dialyzed against 0.15 M NaCl and 0.001 M EDTA, pH 7.0, at 4°C for 24 h.

De novo apolipoprotein synthesis. The effect of Fe2+-ascorbate on newly synthesized and secreted apolipoprotein A-I (apo A-I), apo A-IV, apo B-48, apo B-100, and apo E was assessed as described previously (25). To first induce apolipoprotein synthesis, cells were incubated apically with unlabeled oleic acid bound to albumin in serum-free medium, 24 h before [35S]methionine incubation. The concentration of the unlabeled lipid was equivalent to the labeled substrate described above. During this time, Fe2+-ascorbate was again added to the apical chamber. After a 24-h incubation, cells as well as the outer chambers were rinsed twice with PBS (GIBCO). The apical compartment was replaced with 1.5 ml of methionine-free medium containing the unlabeled substrate and [35S]methionine (100 µCi/ml) (Amersham Life Sciences, 50 mCi/mmol). After incubation for 3 h at 37°C with 5% CO2, the medium from the basolateral compartment was collected. Cells were scraped off the inserts in the cell lysis buffer, as described above. The medium and cell lysates were supplemented with the antiprotease cocktail. To assay a considerable amount of de novo apolipoprotein synthesis, the material from two wells was pooled.

Immunoprecipitation of apolipoproteins. The medium and cell lysates were first supplemented with unlabeled methionine to act as a carrier (final concentration, 0.1 mM). Immunoprecipitation was performed in the presence of excess polyclonal antibodies to human apolipoproteins (Boehringer Mannheim) at 4°C overnight (25, 26). Samples were then washed with Nonidet P-40 (0.05%). They were subsequently centrifuged and resuspended in sample buffer (1.2% SDS, 12% glycerol, 60 mM Tris, pH 7.3, 1.2% beta -mercaptoethanol, and 0.003% bromophenol blue) and analyzed by a linear 4-15% polyacrylamide gradient preceded by a 3% stacking gel, as described previously. Radioactive molecular weight standards (Amersham Life Sciences) were run in the same conditions. Gels were sectioned into 2-mm slices and counted after an overnight incubation with 1 ml of Beckman tissue solubilizer (0.5 N quaternary ammonium hydroxide in toluene) and 10 ml of liquid scintillation fluid (Ready Organic, Beckman). Results for each apolipoprotein studied were expressed as percent dpm/mg protein to assess the specific effect of Fe2+-ascorbate on apolipoprotein synthesis and secretion.

Preparation of microsomes. Microsome fractions were prepared as previously described (22). Briefly, Caco-2 cells were incubated with Fe2+-ascorbate for 24 h and then rinsed, homogenized, and centrifuged for 10 min at 10,000 g at 4°C. The supernatant fraction was centrifuged for 60 min at 100,000 g at 4°C. The washed microsomal pellets were quick frozen and stored at -80°C for later use.

Assay of microsomal HMG-CoA reductase activity. Microsomal enzymatic activity was assayed as described previously (6, 22). The reaction mixture contained 100 mmol/l potassium phosphate (pH 7.4), 150 µg microsomal protein, 20 mmol/l glucose-6-phosphate, 12.5 mmol/l dithiothreitol, 2.5 M NADP, and 1.2 U glucose-6-phosphate dehydrogenase. The reaction was initiated by the addition of [3-14C]-3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) (12,000 dpm/nmol). After incubation for 30 min at 37°C, the [14C]mevalonate formed was converted into lactone, isolated by TLC, and counted using an internal standard to correct for incomplete recovery. HMG-CoA reductase activity was expressed as picomoles of mevalonate synthesized per milligram of protein per minute.

Microsomal ACAT activity. The standard acyl-CoA:cholesterol acyltransferase (ACAT) determination was based on our previous assay (6, 22). We added 5 nmol [14C]oleoyl CoA (sp act, ~10,000 dpm/nmol) to the mixture containing 150 µg microsomal protein to initiate the reaction in a buffer solution (pH 7.5) consisting of cholesterol, 0.04 mol/l KH2PO4, 50 mmol/l NaF, 0.25 mol/l sucrose, and 1 mmol/l EDTA. After incubation for 10 min at 37°C, the reaction was stopped by adding chloroform/methanol (2:1, vol/vol) followed by [3H]cholesteryl oleate as an internal standard to estimate recovery.

Statistical analysis. All values were expressed as means ± SE. Data were analyzed by two-tailed Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MDA generation after Fe2+-ascorbate exposure. The effectiveness of Fe2+-ascorbate in initiating lipid peroxidation was tested after incubation with Caco-2 cells. At the end of a 24-h culture period, the degree of lipid peroxidation was determined by measuring MDA in medium and cells. As illustrated in Fig. 1, Fe2+-ascorbate promotes the production of peroxidation above baseline values, and the formation of MDA increased with rising Fe2+-ascorbate concentrations in a dose-dependent manner. Neither Fe2+ nor ascorbate alone could induce lipid peroxidation (data not shown). The concentration-response relationship for intestinal epithelial peroxidation was 10-fold higher in the apical medium than in cells.


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Fig. 1.   Lipid peroxidation in Caco-2 cells challenged with Fe2+-ascorbate (1:10). Caco-2 cells were incubated with increasing concentrations of Fe2+-ascorbate for 24 h at 37°C. Iron concentrations shown on x-axis represent actual Fe2+ concentration along with a corresponding 10-fold higher ascorbate concentration at each Fe2+ amount. A concentration-dependent increase in equivalent malondialdehyde (MDA) formed was observed between 50 and 400 µM of Fe2+ in cells (A) and medium (B). Values are means ± SE for 2 different experiments.

Effect of Fe2+-ascorbate on Caco-2 cell functional integrity. Caco-2 cell integrity and viability were assessed by sucrase activity, cell monolayer transepithelial resistance, DNA and protein content, and trypan blue exclusion after an incubation of 24 h. As shown in Fig. 2, transepithelial resistance and sucrase activity remained unchanged with increasing concentrations of Fe2+-ascorbate. Similarly, the cell DNA and protein content was unaffected by the addition of the oxygen-radical generating system. At all the Fe2+-ascorbate concentrations tested, the cell protein-to-DNA ratio did not show any significant variability. Cell viability by trypan blue exclusion was also assessed and was uniformly >90% in the absence or presence of Fe2+-ascorbate.


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Fig. 2.   Effect of increasing concentrations of Fe2+-ascorbate on transepithelial resistance (A), sucrase activity (B), cell protein content (C), and cell DNA content (D). Values are means ± SE for 2 separate preparations.

Effect of Fe2+-ascorbate on lipid synthesis and secretion. Lipid synthesis and secretion were determined in the presence and absence of Fe2+-ascorbate after a 24-h incubation period. When oleic acid bound to BSA was presented to confluent Caco-2 cell monolayers, the total fatty acid incorporation was decreased in cells with Fe2+-ascorbate (596,415 ± 9,960 dpm/mg protein) compared with cells without Fe2+-ascorbate (698,208 ± 15,084 dpm/mg protein). A similar trend was also noted in the medium (18,436 ± 907 vs. 24,206 ± 580 dpm/mg protein for cells with or without Fe2+-ascorbate, respectively). This decrease was essentially accounted for by a reduction in TG (15%, P < 0.0002 in cells; 26%, P < 0.0001 in medium) and PL (16%, P < 0.0005 in cells; 17%, P < 0.03 in medium) (Fig. 3). In the second step of our studies, the effect of Fe2+-ascorbate on Caco-2 cell cholesterol biogenesis was evaluated using [14C]acetate. As can be seen in Fig. 4, Fe2+-ascorbate significantly increased the incorporation of the radiolabeled precursor into total cholesterol. The raised level of total cholesterol (96%, P < 0.0001 in cells; 142%, P < 0.03 in medium) was attributable to the increase of both fractions of FC (105%, P < 0.0001) and CE (67%, P < 0.0001) in Caco-2 cells and to FC (193%, P < 0.03) only in the medium.


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Fig. 3.   Effect of Fe2+-ascorbate on Caco-2 cell lipid synthesis and secretion. Fe2+-ascorbate was incubated in apical compartment for 24 h, with [14C]oleic acid as substrate. Cells were harvested and basolateral medium was collected for de novo cell (A) and medium (B) lipid synthesis as described in MATERIALS AND METHODS. Values are means ± SE for 6 different experiments. PL, phospholipids; TG, triglycerides; CE, cholesteryl ester. a P < 0.03; b P < 0.0005; c P < 0.0002; d P < 0.0001.



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Fig. 4.   Effect of Fe2+-ascorbate on Caco-2 cell cholesterol synthesis and secretion. Fe2+-ascorbate was incubated in the apical compartment for 24 h, with [14C] acetate as a precursor. Cells were harvested and basolateral medium was collected for de novo cell (A) and medium (B) cholesterol synthesis as described in MATERIALS AND METHODS. Values are means ± SE for 6 different experiments. TC, total cholesterol; FC, free cholesterol. aP < 0.03; bP < 0.0001.

Effect of Fe2+-ascorbate on microsomal sterol enzymes. In view of the impact of lipid peroxidation on cholesterol biogenesis, we determined the effect of Fe2+-ascorbate on HMG-CoA reductase (the rate-limiting enzyme in cholesterol synthesis) and ACAT (the enzyme responsible for the acylation of cholesterol into CE). The incubation of microsomal preparations from Caco-2 cells with Fe2+-ascorbate caused a marked elevation in HMG-CoA reductase activity (70%, P < 0.04) without altering ACAT activity (Fig. 5).


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Fig. 5.   Influence of Fe2+-ascorbate on key regulatory enzymes. Caco-2 cells were incubated with Fe2+-ascorbate for 24 h at 37°C. Thereafter, microsomes were isolated and assayed for DL-3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and acyl-CoA:cholesterol acyltransferase (ACAT) as described in MATERIALS AND METHODS. a P < 0.04.

Effect of Fe2+-ascorbate on apolipoprotein synthesis. One of the major objectives of these studies was to examine the modulation of apolipoprotein elaboration by Fe2+-ascorbate. The treatment with the oxidant system resulted in a significantly reduced yield of the main apolipoproteins normally synthesized by the intestine: A-I (18%, P < 0.05), A-IV (38%, P < 0.05), and B-48 (45%, P < 0.03) (Fig. 6). Fe2+-ascorbate also affected the exocytosis of apo B-100 (35%, P < 0.02), apo B-48 (36%, P < 0.003), and apo A-IV (58%, P < 0.0008).


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Fig. 6.   Effect of Fe2+-ascorbate on Caco-2 cell apolipoprotein synthesis. Fe2+-ascorbate was incubated in apical compartment for 20 h with unlabeled oleic acid to stimulate apolipoprotein biogenesis. Apolipoproteins A-I, A-IV, B-100, B-48, and E were analyzed after a 3-h incubation with [35S]methionine by SDS gel electrophoresis. Values, relative to cells (A) and medium (B), represent means ± SE for 3 separate experiments. Results are expressed as dpm/mg cellular protein. a P < 0.05; b P < 0.03; c P < 0.02; d P < 0.0003; e P < 0.0008.

Effect of Fe2+-ascorbate on lipoprotein secretion. As expected from the inhibition of lipid synthesis and apolipoprotein biogenesis, Fe2+-ascorbate affected the secretion of all four classes of lipoproteins studied (Fig. 7). A significant reduction was noted in chylomicrons (50%, P < 0.0001), VLDL (40%, P < 0.0001), LDL (37%, P < 0.0001), and HDL (30%, P < 0.0001) as shown in Fig. 7.


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Fig. 7.   Effect of Fe2+-ascorbate on Caco-2 cell lipoprotein secretion. Fe2+-ascorbate was incubated in the apical compartment for 20 h, with [14C]oleic acid as a precursor. Cells were harvested and basolateral medium was collected for lipoprotein isolation as described in MATERIALS AND METHODS. Values are means ± SE for 6 separate experiments. CM, chylomicrons; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL, high-density lipoprotein. a P < 0.0001.

Effect of BHT. To uncover whether lipid peroxidation was fully behind the aforementioned lipid and lipoprotein alterations, Caco-2 cells were cultured in the presence of BHT before incubation with Fe2+-ascorbate. As noted in Table 1, the preincubation of Caco-2 cells with BHT led to a protection against Fe2+-ascorbate-mediated lipid peroxidation. Indeed, BHT in contrast to other antioxidants tested, such as vitamin E and glutathione (results not shown), was effective in reducing MDA generation. Concomitantly, BHT improved lipid esterification, cholesterol synthesis, and CM formation.

                              
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Table 1.   Influence of BHT on Fe2+-ascorbate-mediated lipid derangements


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Caco-2 cell line has been used to examine a variety of intestinal functions, including nutrient absorption (24). This intestinal model forms a highly polarized monolayer, exhibits many of the features of small intestinal cells, and displays vectorial transport. Because Caco-2 cells reproduce several of the normal physiological responses to various modulatory agents, we examined their usefulness to study the effect of Fe2+-ascorbate-induced lipid peroxidation on the intracellular phase of lipid absorption, i.e., intraenterocyte esterification and resynthesis of lipolytic products, the biogenesis of apolipoproteins required for TG-rich particles, and lipoprotein secretion. Our results show for the first time that Fe2+-ascorbate can clearly affect the efficiency of Caco-2 cells to assemble and transport lipids in lipoprotein forms.

Iron occupies a central role in oxygen-radical chemistry, because it can initiate oxygen radical formation (9). Not only is iron a catalyst in the Haber-Weiss reaction, but it is also involved in the initiation and propagation of lipid peroxidation (14). Although the mechanisms underlying the cytotoxicity of iron in different organs are not completely understood, many reports have pointed to the participation of iron-mediated peroxidation in numerous pathological states, including atherosclerosis (38), cancer (16), ischemia-reperfusion injury (10), inflammatory bowel disease (35), and conditions of iron overload (4). Several laboratories (3, 5, 19) have shown the ability of Fe2+ to initiate strong lipid peroxidation, whereas ascorbic acid can amplify the oxidative potential of iron by promoting metal ion-induced lipid peroxidation. The data presented here clearly indicate that the Fe2+-ascorbate system functioned as a producer of lipid peroxidation and, at the same time, altered the integrity of intracellular fat transport. It is noteworthy that the iron dose used in the current study is comparable to normal iron concentration in the gut (2). The deteriorations resulting from the exposure of Caco-2 cells to Fe2+-ascorbate are probably attributable to oxidative stress, because the addition of the BHT antioxidant simultaneously prevented the occurrence of lipid peroxidation and improved the cellular processes of lipid absorption. Previous studies (19) have addressed the issue of glucose transport under the influence of in vitro peroxidation, employing guinea pig brush-border membrane vesicles. The peroxidative attack, initiated by Fe2+-ascorbate, resulted in the reduction of sodium-dependent glucose transport (19). All these data, taken together, strongly support the causative derangement of nutrient transport by oxidative stress.

Numerous studies have already emphasized that essential fatty acids are absolutely necessary for 1) the control of microviscosity and membrane fluidity of most cells (3), 2) the regulation of membrane protein (5), and 3) the synthesis of eicosanoids, such as prostaglandins, leukotrienes, and related substances that have a profound influence on many transport processes (23, 40). Our understanding of how lipid peroxidation influences the intestinal phase, i.e., the formation of lipid-carrying lipoproteins, remains sketchy. Nevertheless, it is tempting to assume that a peroxidative attack on membranes could deplete essential fatty acid content, thereby modifying intracellular fat transport. Accordingly, our previous work (23) stressed the critical role of essential fatty acid deficiency in the biophysical and biochemical events involved in fat absorption. The peroxidative modification of unsaturated phospholipids in the endoplasmic reticulum (the site of lipid esterification, apolipoprotein synthesis, and lipoprotein assembly) could conceivably hamper enterocyte lipid transport. Ongoing exploration of the mechanisms involved will have an important bearing on our understanding of disease states associated with peroxidative stress, such as inflammatory bowel diseases (35). In particular, Crohn's disease is known for its overproduction of harmful oxidants, which have been implicated in the pathophysiology of chronic inflammation and intestinal injury. Peroxidative damage to the absorptive epithelial cells is likely a major contributor to impaired transport of electrolytes, trace metals, and vitamins as well as other micronutrients. Given the negative influence of both cytokines (32) and essential fatty acid deficiency (23) on fat absorption, our data suggest that the combination of excessive proinflammatory cytokines and lipid peroxidation may give rise to disturbances in epithelial absorptive processes in inflammatory bowel diseases, such as impaired lipid transport. However, studies have not yet clearly identified whether essential fatty acid deficiency and lipid soluble vitamin depletion are the result of malabsorption vs. inadequate intake to meet needs.

We (6) previously determined the effect of iron overload on plasma lipid profile and lipoprotein composition in rats administered a diet enriched with 3% iron carbonyl. The latter dietary regimen provoked marked hyperlipidemia, evidenced by the concomitant increase in plasma TG and cholesterol. In addition, various chemical abnormalities characterized the composition and size of lipoproteins. These disturbances were correlated with lipid peroxidation as reflected by elevated MDA concentrations. However, in the current study, Caco-2 cells submitted to lipid peroxidation displayed defective lipoprotein production. In view of this compromised intestine lipoprotein secretion, it is therefore tempting to suggest that the in vivo hyperlipoproteinemia identified in rats with iron loading is attributable to liver hypersecretion only. Additional work is needed to clarify this important issue.

The experiments performed in the current study were aimed at defining whether iron-catalyzed lipid peroxidation could modify intestinal cholesterol metabolism. Our data revealed that de novo cholesterogenesis, assessed by the incorporation of [14C]acetate, was markedly increased in treated Caco-2 cells. This was associated with a significant enhancement of HMG-CoA reductase activity. At this time, we are not able to specify whether iron-catalyzed lipid peroxidation modulates the enzyme activity by altering its concentration through transcriptional or posttranscriptional modifications. Iron-catalyzed lipid peroxidation also may attack polyunsaturated fatty acids, resulting in changes in the physical properties of the fluidity of the membrane in which HMG-CoA reductase is embedded. Such indirect effects have been shown to alter the immediate environment of the enzyme, thereby affecting its function (11, 41).

Unlike HMG-CoA reductase, ACAT activity was resistant to iron-induced lipid peroxidation. Furthermore, concurrent to iron-catalyzed peroxidation in the livers of rats with iron overload, the activities of the two enzymes were dissimilar; HMG-CoA reductase was suppressed while ACAT was activated (6). Apparently, the two enzymes respond to peroxidative stress differently.

Apolipoproteins are necessary for exogenous and endogenous lipid transport. Their biosynthesis is a principal determinant of plasma lipoprotein levels, and defects in their synthesis or function affect lipoprotein metabolism (21). Our study showed that iron-catalyzed lipid peroxidation resulted in diminished newly synthesized apolipoproteins. Even the biogenesis of apo B that is crucial for the assembly and exocytosis of TG-rich lipoprotein was markedly impaired. At present, the mechanisms involved in these abnormalities are not elucidated, including apo B elongation or translation, the rate of apo B translocation across the endoplasmic reticulum, and intracellular degradative pathway of apo B (18). Similarly, Murthy et al. (33) found that less newly synthesized apo B was secreted by cells incubated with 13-hydroxyoctadecadienoic acid, an oxidized lipid. Murthy et al. (33) suggested that defective apo B translocation was responsible for the reduced apo B secretion. Furthermore, the original observation that the diminished synthesis of apo A-IV, apo B, and lipoprotein caused by Fe2+-ascorbate could be attributed to its direct effects on TG production is unlikely. The incubation of Caco-2 cells with Fe2+-ascorbate in the absence of oleic acid, the main precursor of TG biogenesis, resulted in similar differences between control and treated cells (results not shown).

In summary, the Fe2+-ascorbate system appeared to be very effective in promoting lipid peroxidation of Caco-2 cells in view of the markedly increased MDA levels. Concomitantly, it elicited a reduction in lipid esterification and synthesis, apolipoprotein biogenesis, and lipoprotein secretion. We conclude that long-lasting lipid peroxidation may overwhelm intraluminal antioxidant defense and impair intestinal fat transport.


    ACKNOWLEDGEMENTS

We thank Danielle St-Cyr Huot for typing the manuscript.


    FOOTNOTES

This study was supported by grants from the Medical Research Council of Canada (MT-10584) and the Canadian Foundation for Crohn's and Colitis.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: E. Levy, Gastroenterology Unit, Hôpital Sainte-Justine, 3175 Côte Ste-Catherine Rd., Montreal, Quebec, Canada H3T 1C5 (E-mail: levye{at}justine.umontreal.ca).

Received 30 July 1999; accepted in final form 4 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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