Departments of 1 Physiology and Pharmacology and 2 Pathobiology, Texas A&M University, Texas Veterinary Medical Center, College Station, Texas 77843-4466
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ABSTRACT |
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High levels of saturated,
branched-chain fatty acids are deleterious to cells and animals,
resulting in lipid accumulation and cytotoxicity. Although fatty acid
binding proteins (FABPs) are thought to be protective, this hypothesis
has not previously been examined. Phytanic acid (branched chain,
16-carbon backbone) induced lipid accumulation in L cell fibroblasts
similar to that observed with palmitic acid (unbranched,
C16): triacylglycerol free fatty acid > cholesterol > cholesteryl ester
phospholipid. Although
expression of sterol carrier protein (SCP)-2, SCP-x, or liver FABP
(L-FABP) in transfected L cells reduced [3H]phytanic acid
uptake (57-87%) and lipid accumulation (21-27%), nevertheless [3H]phytanic acid oxidation was inhibited
(74-100%) and phytanic acid toxicity was enhanced in the order
L-FABP
SCP-x > SCP-2. These effects differed markedly from
those of [3H]palmitic acid, whose uptake, oxidation, and
induction of lipid accumulation were not reduced by L-FABP, SCP-2, or
SCP-x expression. Furthermore, these proteins did not enhance the
cytotoxicity of palmitic acid. In summary, intracellular FABPs reduce
lipid accumulation induced by high levels of branched-chain but not
straight-chain saturated fatty acids. These beneficial effects were
offset by inhibition of branched-chain fatty acid oxidation that
correlated with the enhanced toxicity of high levels of branched-chain
fatty acid.
sterol carrier protein-2; sterol carrier protein-x; liver fatty acid binding protein; fatty acids; phytanic acid; palmitic acid; lipid mass; toxicity
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INTRODUCTION |
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ALTHOUGH HIGH LEVELS of branched-chain (6, 52, 64, 94, 95, 97) or straight-chain (8, 14, 29, 35, 49, 66, 86, 87, 92, 101) saturated fatty acids induce lipid accumulation and toxicity in cultured cells and animals, the potential role of intracellular fatty acid binding proteins in modulating these effects is unknown. The existing data do not allow a priori prediction of whether the intracellular fatty acid binding proteins may be protective.
An early hypothesis is that intracellular fatty acid binding proteins bind fatty acids in the cytoplasm and thereby prevent their nonspecific detergent effects on disrupting membrane functions (61). Indeed, liver fatty acid binding protein (L-FABP) (57) and sterol carrier protein (SCP)-2 (77) have high affinity [dissociation constant (Kd) in nM range] for both branched-chain and straight-chain fatty acids. L-FABP and SCP-2 also bind branched- and straight-chain saturated fatty acyl CoA esters (20, 33, 81, 88) with high affinity (Kd in nM range). Furthermore, intracellular fatty acid binding protein levels are in the 0.2-0.4 mM range in tissues (liver, intestine, heart) active in fatty acid metabolism. However, if fatty acids were not rapidly metabolized, then intracellular fatty acid binding proteins would quickly become saturated with ligand and would not protect against toxic effects of fatty acids. On the contrary, because of the rapid metabolism of fatty acids, the intracellular free fatty acid (reviewed in Ref. 47) and fatty acyl CoA (reviewed in Refs. 18, 28) concentrations are normally maintained at constant, very low levels. Thus it is unlikely that fatty acid binding proteins would be protective simply by binding fatty acids.
A considerable body of evidence suggests that intracellular fatty acid binding proteins actually enhance, rather than prevent, the formation of lipids that are typically induced by high levels of fatty acids. However, these experiments were performed under conditions of low fatty acid concentrations. L-FABP, intestinal fatty acid binding protein (I-FABP), and SCP-2 stimulate microsomal incorporation of fatty acyl CoAs into glycerides in vitro (10, 38-40, 76, 85) and in transfected cells (3, 47, 85). Both SCP-2 and L-FABP also stimulate microsomal incorporation of fatty acyl CoA into cholesteryl esters in vitro (12, 51, 69, 70, 91) and in transfected cells (36, 37, 56). Thus the data obtained with low concentrations of fatty acids indicate that fatty acid binding proteins are likely to induce rather than inhibit accumulation of fatty acids in esterified lipids. However, the effect of intracellular fatty acid binding proteins on lipid accumulation and toxicity in the presence of high fatty acid levels is not known. In fact, some data suggest that induction of lipid accumulation may not necessarily be coupled to toxicity of the fatty acid (25, 26).
Although this has not been demonstrated directly in intact cells, in
vitro experiments are consistent with a role for intracellular fatty
acid binding proteins in enhancing oxidation of low levels of fatty
acids. Branched-chain saturated fatty acids exclusively undergo
-oxidation and partial
-oxidation in peroxisomes (reviewed in
Ref. 94). SCP-2 and, even more so, SCP-x are present in
highest concentrations in peroxisomes (4, 23, 59, 71).
Importantly, SCP-x is a peroxisomal enzyme that exclusively exhibits
3-ketoacyl-CoA thiolase activity with branched-chain substrates
(1, 2, 96). In contrast, straight-chain fatty acids are
-oxidized primarily in mitochondria (32). Fatty acid
binding proteins enhance transport of saturated fatty acids in intact
cells (45, 47, 53, 54, 100). In addition, fatty acid
binding proteins enhance transport of fatty acyl CoA to isolated
mitochondria (63) and stimulate their mitochondrial
-oxidation (62, 65) in vitro. Low levels of L-FABP are
associated with the outer envelope of mitochondria (10)
and SCP-2 has also been detected in mitochondria (23, 24,
41). Although these in vitro data are consistent with a role for
the intracellular fatty acid binding proteins in oxidation of low
levels of fatty acids, their effect in the presence of high levels of
fatty acids remains to be determined.
Finally, studies with gene-targeted mice provide additional evidence for fatty acid binding protein functioning in the oxidation of low levels of branched-chain and straight-chain fatty acids. For example, disruption of the heart fatty acid binding protein (H-FABP) gene in mice results in 90% inhibition of fatty acid oxidation by cardiac myocytes exposed to low levels of straight-chain saturated fatty acid (i.e., palmitic acid) (9, 67). Branched-chain saturated fatty acids such as phytanic acid accumulate in sera of SCP-x/SCP-2 gene-ablated mice fed normal chow or high levels of phytol (81). Although high levels of phytol are more toxic in SCP-x/SCP-2 gene-ablated mice, lipid accumulation is reduced rather than increased as expected (81). Although the latter studies represent the first report on the effect of fatty acid binding proteins on lipid accumulation and toxicity of high levels of branched-chain fatty acids, it must be noted that multiple fatty acid binding proteins are missing (i.e., both SCP-x and SCP-2) and, concomitantly, L-FABP is upregulated fourfold in these gene-targeted mice (81). Thus the individual contributions of SCP-x, SCP-2, and L-FABP in modifying the lipid accumulation and toxicity induced by high levels of branched-chain, much less straight-chain, saturated fatty acids remain to be resolved.
The present investigation used transfected L cell fibroblasts individually overexpressing L-FABP, SCP-2, or SCP-x to address the hypothesis that these proteins protect the cell from lipid accumulation and toxicity induced by branched- and straight-chain saturated fatty acids. Phytanic acid (16-carbon backbone with 4 methyl branches) was chosen as a model because it is the most prevalent dietary branched-chain saturated fatty acid. Furthermore, phytanic acid has the same carbon chain length as the unbranched saturated palmitic acid, one of the most prevalent dietary straight-chain saturated fatty acids.
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MATERIALS AND METHODS |
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Materials. Calcein AM and ethidium homodimer-1 were purchased from Molecular Probes (Eugene, OR); phytanic acid, palmitic acid, bovine serum albumin (BSA; essentially fatty acid free), and fetal bovine serum (FBS) were obtained from Sigma (St. Louis, MO); [2,3-3H]phytanic acid (specific activity of 50 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA); [9,10-3H]palmitic acid (specific activity of 36.3 Ci/mmol) was purchased from Dupont New England Nuclear (Boston, MA); silica gel G plates were purchased from Analtek (Newark, DE); neutral lipid and fatty acid standards were purchased from Nu-Chek Prep (Elysian, MN); and phospholipid standards were purchased from Avanti (Alabaster, AL). Rabbit polyclonal antisera to recombinant human SCP-2, mouse SCP-x, mouse acyl CoA binding protein (ACBP), and rat L-FABP were prepared as described previously (4). Antiserum against CD-36 (also called fatty acid translocase; FAT) was purchased from Research Diagnostics (Flanders, NJ). Rabbit polyclonal anti-fatty acid transport protein (FATP) was a generous gift from Dr. J. Schaffer (Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO). Sheep anti-bovine catalase was purchased from Biodesign (Kennebunk, ME). Rabbit anti-mouse peroxisomal membrane protein-70 (PMP-70) was purchased from Affinity Bioreagents (Golden, CO). All reagents and solvents used were of the highest grade available and were cell culture tested.
L cell fibroblast culture.
Murine L cells (L arpttk
) were grown to
confluence at 37°C and 5% CO2 in Higuchi medium
(31) supplemented with 10% FBS (Hyclone, Logan, UT). The
cell lines used were derived as follows: the mock-transfected cells
(designated as control) (37, 50) and cells overexpressing L-FABP (4, 37) were developed as described previously.
Because the SCP-x/SCP-2 gene encodes for two proteins (15-kDa pro-SCP-2 and 58-kDa SCP-x), it was necessary to prepare clones transfected with
the cDNAs separately encoding the 15-kDa pro-SCP-2 and the 58-kDa
SCP-x.
Presence of mitochondria and peroxisomes in L cells. The presence of mitochondria and peroxisomes was determined by Western blotting and/or electron microscopy. L cells were homogenized, and fractions enriched in organelles involved in fatty acid oxidation, i.e., mitochondria and peroxisomes, were obtained by differential centrifugation and discontinuous sucrose gradient centrifugation as described previously (24). A 12,000 g pellet from L cell homogenates was run over a sucrose gradient to produce two bands (24): one at 37% sucrose containing both mitochondria and peroxisomes and one at 33% sucrose identified by Western blot analysis as consisting of mostly peroxisomes (see RESULTS). L cell homogenates and peroxisomal enriched fractions were resolved with tricine SDS-PAGE gels (16%), transferred to nitrocellulose membranes, and probed against affinity-purified antisera against peroxisomal markers PMP-70 and catalase basically as described previously (68). The peroxisome-enriched bands were also collected and processed for electron microscopy as described previously (7).
Supplementation of L cells with branched- and straight-chain saturated fatty acids: preparation of fatty acid-BSA complexes. Fatty acid-BSA complexes were prepared with the method of Spector and Hoak (84), in which palmitic acid (dissolved in hexane) and phytanic acid (dissolved in dimethylformamide; DMF) were added to celite particles (Sigma, St. Louis, MO) with 1 µmol of fatty acid per 10 mg of solid phase. After the solvent was removed under a N2 stream, the fatty acid-coated particles were mixed with lipid-free albumin in serum-free medium for 45 min at room temperature. Celite particles were removed by centrifugation, and the resultant supernatant was adjusted to pH 7.4. The concentration of fatty acid and BSA in the complexes was determined by the methods of Dole and Meinertz (15) and Bradford (11), respectively.
Cells from the respective cell lines were plated at ~1.4 × 106 cells/10-cm dish in serum-containing medium. After 24 h, the cells were washed extensively with PBS and the medium was changed to serum-free medium to limit the presence of serum-derived fatty acids. BSA or palmitic acid-BSA or phytanic acid-BSA complexes (25 µM fatty acid equivalents, ~90 µM BSA) were then added to the cells cultured in serum-free medium followed by incubation for 6 h at 37°C before lipid extraction. These concentrations of fatty acid-BSA complexes were similar to those found in the serum-containing medium used to culture cells (42, 60), and the ratios of fatty acid to BSA in the complexes were similar to those reported previously (15). Under these conditions not only was cell viability >95%, but both palmitic acid-BSA and phytanic-BSA complexes induced lipid accumulation in all three cell lines (see RESULTS).Measurement of lipid accumulation and analysis. Cells incubated with BSA or fatty acid-BSA complexes as described above were extracted with n-hexane-2-propanol (3:2 vol/vol) (30, 37). Lipid classes were resolved on silica gel G thin-layer chromatography plates developed in petroleum ether-diethyl ether-methanol-acetic acid (90:7:2:0.5 vol/vol/vol/vol) (30, 37). Total cholesterol, free fatty acid, triacylglyceride, and cholesteryl ester content were determined by the method of Marzo et al. (46). Total phospholipid content was determined by digesting the phospholipid fraction in deionized water and perchloric acid for 1 h at 180°C followed by addition of ammonium molybdate and ascorbic acid. After further heating for 5 min in a boiling water bath, the sample was cooled and the absorbance was read at 797 nm to quantify total lipid phosphorus. Lipids were stored under an atmosphere of N2 to limit oxidation. All glassware was washed with sulfuric acid-chromate before use. Protein concentration was determined from the dried protein extract residue digested overnight in 0.2 M KOH.
Uptake of radiolabeled fatty acids by cultured L cells. To determine the extent of fatty acid uptake, cells were incubated with BSA-fatty acid complexes (25 µM fatty acid equivalents, ~90 µM BSA) in the presence of trace amounts of [3H]palmitic acid or [3H]phytanic acid (2 µCi/35-mm dish) for 6 h at 37°C. The medium was then removed, and lipids were extracted from the medium and from the cells as described previously (19). The residual, nonlipid residue (remaining after lipid extraction of the cell monolayer) was dissolved in 0.2 M KOH overnight and assayed for protein and radiolabel content. Radiolabeled lipids were quantified in a liquid scintillation cocktail (Scinti Verse, Fisher Scientific, Pittsburgh, PA) on a Packard 1600TR liquid scintillation counter (Meridian, CT).
Oxidation of radiolabeled fatty acids by cultured L cells. To determine the extent of fatty acid oxidation, cells were incubated with fatty acid-BSA complexes (25 µM fatty acid equivalents, ~90 µM BSA) in the presence of trace amounts of [3H]palmitic acid and [3H]phytanic acid (2 µCi/35-mm dish) for 6 h at 37°C. Oxidation was measured from the formation of water-soluble tritiated degradation products released into the culture medium after the medium was removed and extracted by the method of Folch et al. (19) with modifications as described previously. Radioactivity was quantified in a liquid scintillation cocktail (Scinti Verse) on a Packard 1600TR liquid scintillation counter.
Determination of saturated fatty acid cytotoxicity in L cells: flow cytometry. To determine the cytotoxicity of saturated fatty acids, an assay kit (Molecular Probes) composed of calcein AM and ethidium homodimer-1 was used. The assay was based on membrane-permeant calcein AM, which, after cleavage by intracellular esterases, yielded a green cytoplasmic fluorescence (with a blank nucleus) in live cells. Ethidium homodimer-1 was used as a membrane-impermeant fluorophore that penetrated membrane-compromised (dead) cells to label nucleic acids in the nucleus and yield a red fluorescence. By using both dyes simultaneously, the ratio of live to dead cells was detected and quantitated by flow cytometry. Briefly, cells were washed with PBS to remove serum before addition of the fatty acid-BSA complexes. At various timed intervals (from 12 to 96 h) the cells were prepared for analysis by removing the medium from each plate and washing once with PBS, followed by incubation in trypsin (0.25%, 1 mM EDTA-4Na). After 5 min, trypsinization was stopped by addition of complete medium containing 10% FBS, and the cell count was determined. The trypsinized cell suspensions and all culture medium and PBS washes removed from the dishes were combined by centrifuging at 1,000 rpm for 6 min, and the resulting cell pellets were then resuspended in Puck's buffer (in mM: 1 Na2HPO4, 0.9 H2PO4, 5.0 KCl, 1.8 CaCl2, 0.6 MgSO4, 6 glucose, 138 NaCl, and 10 HEPES) to give a cell density of ~9 × 104 cells/ml. To limit artifacts associated with potential toxicity and/or saturation of live/dead dyes in the assay, dye concentrations were individually titered over a broad range to obtain an optimal dye solution: 0.1 µM calcein AM-0.8 µM ethidium homodimer-1. This dye solution was added to the cell suspension and incubated as indicated in the assay kit. Each sample was analyzed with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) at a low flow rate (~670 cells/s) and simultaneous excitation of both dyes at 488 nm. Fluorescence emission was detected through two separate filters, calcein (green fluorescence at 530 nm) and ethidium homodimer-1 (red fluorescence at 585 nm). As a result, the live and dead cells were resolved into two bivariate populations by flow cytometry.
Measurement of plasma membrane fatty acid transport proteins and intracellular acyl CoA binding protein. Cell homogenates of control and SCP-2-, SCP-x-, and L FABP-expressing cells were analyzed by Western blot analysis to determine whether expression levels of plasma membrane proteins associated with fatty acid transport were altered. Cell homogenates (10 µg) were run on tricine gels (16%) before transferral to 0.45-µm nitrocellulose paper (Sigma) by electroblotting in a continuous buffer system at 0.8 mA/cm2 for 2 h. After transfer, blots were treated as described previously (4) with affinity-purified antisera against the major plasma membrane fatty acid transport proteins: CD-36 (FAT) and FATP. To determine whether supplementation with fatty acid-BSA complexes altered the expression of proteins associated with fatty acid uptake and transport, Western blot analysis was also performed on samples supplemented with BSA, palmitic-BSA, and phytanic acid-BSA. Cell homogenates were treated as described above with the blots probed against affinity-purified antisera to CD-36 (FAT) and FATP. Proteins were quantified by densitometric analysis after image acquisition with a single-chip charge-coupled device (CCD) video camera and a computer workstation (IS-500 system; Alpha Innotech, San Leandro, Ca). Image files were analyzed (mean 8-bit gray scale density) with NIH Image (available by anonymous FTP).
L cells also contain high levels of another intracellular protein, ACBP (27, 28), that may indirectly play a role in fatty acid metabolism by enhancing fatty acyl CoA transport and/or utilization by oxidative organelles (reviewed in Refs. 18, 28). Therefore, the above experiments were repeated by separately probing Western blots with affinity-purified antisera against ACBP.Statistics. All values are expressed as means ± SE with n and P indicated in RESULTS. Statistical analysis was performed with analysis of variance (ANOVA) combined with Dunnett's multiple-comparison test (GraphPad Software, San Diego, CA). Values with P < 0.05 were considered statistically significant.
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RESULTS |
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Expression of L-FABP, SCP-2, and SCP-x in transfected L cell
fibroblasts.
Expression of L-FABP, SCP-2, and SCP-x in transfected L cell
fibroblasts was determined by Western blot analysis of cell homogenates probed with specific antisera to the proteins as described in MATERIALS AND METHODS. Although levels of L-FABP, SCP-2, or
SCP-x were not detected in control mock-transfected cells, quantitative analysis of the transfected clones indicated that protein expression levels were 0.4% (Fig. 1A,
lane 2), 0.036% (Fig. 1B, lane 2), and 0.01% (Fig. 1C, lane 3) of soluble protein,
respectively, consistent with earlier findings. Furthermore, partial
posttranslational cleavage of the 58-kDa SCP-x protein resulted in
detectable 43-kDa protein and up to a twofold increase in level of
13-kDa SCP-2 (Fig. 1C, lane 3, and Fig.
1B, lane 3, respectively). The partial posttranslational cleavage of 58-kDa SCP-x confirmed previous suggestions (4, 59).
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Presence of fatty acid oxidative organelles in L cells:
mitochondria and peroxisomes.
To confirm that L cell fibroblasts have the basic machinery necessary
for fatty acid oxidation, Western blot analysis was performed on L cell
homogenates and subcellular fractions enriched in peroxisomes and
mitochondria (Fig. 2). The peroxisomal
markers PMP-70 (Fig. 2A, lane 1) and catalase
(Fig. 2B, lane 2) were both present in L cell
homogenates. The level of catalase was about fivefold higher in the
peroxisome-enriched fraction (Fig. 2B, lane 3)
compared with the L cell homogenate (Fig. 2B, lane
2). Electron micrographs of the peroxisome-enriched fraction
showed that L cells contain mitochondria, the primary site of fatty
acid -oxidation (Fig. 2C), as well as peroxisomes, the
site of
- and/or
-oxidation of branched-chain and very long fatty
acids (Fig. 2C).
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Effect of acyl chain branching on saturated fatty acid-induced lipid accumulation in L cell fibroblasts. Although straight-chain saturated fatty acids such as palmitic acid are known to induce lipid accumulation in cultured cells (e.g., L cell fibroblasts) and animals (reviewed in Ref. 26), the effect of fatty acid acyl chain branching on this lipid accumulation is not known. To resolve this issue, control L cells were supplemented with phytanic acid (16-carbon backbone with 4 methyl branches) presented as an albumin complex as described in MATERIALS AND METHODS. For comparison with the effects of straight-chain saturated fatty acid, cells were also supplemented with palmitic acid (16-carbon chain length fatty acid without branching). The cells were incubated with the respective fatty acid-albumin complexes in serum-free medium for 6 h followed by washing, lipid extraction, and analysis as indicated in MATERIALS AND METHODS.
Supplementation of L cells with phytanic acid-BSA complex induced a 1.7-fold increase in total lipid mass (nmol lipid/mg protein) compared with supplementation with BSA only (Table 1). However, this increase was selective among the major lipid classes in the following order: 6.5-fold for triacylglycerol > 3.0-fold for free fatty acid > 2.0-fold for cholesterol > 1.3-fold for cholesteryl ester
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Fatty acid binding protein expression differentially alters
saturated fatty acid-induced lipid accumulation.
To examine the effect of fatty acid binding proteins on protection of
the cells against saturated fatty acid-induced lipid accumulation, the
above experiments were repeated with transfected L cells individually
overexpressing SCP-2, SCP-x, or L-FABP at levels in the range of those
reflecting their respective physiological values. Control,
untransfected L cells did not differ from mock-transfected L cells.
Three types of experiments were performed. First, the effect of SCP-2,
SCP-x, and L-FABP expression on basal (i.e., in the absence of added
palmitic or phytanic acid) lipid mass was examined (Fig.
3). Only SCP-x expression significantly
altered basal lipid mass (nmol lipid/mg protein) to elicit a 1.6-fold decrease (P < 0.01; n = 3-6)
compared with the control cells (Fig. 3). In contrast, expression of
SCP-2 or L-FABP had no effect on basal lipid mass.
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Effect of methyl branching on saturated fatty acid-induced free
(unesterified) fatty acid accumulation in L cells.
As indicated above, cellular levels of fatty acids in esterified form
(e.g., triglycerides, phospholipids, cholesteryl esters) are high and
depend on available dietary fatty acids. In contrast, because of their
potent detergent as well as metabolic regulatory effects, cellular
levels of free (unesterified) fatty acids are more tightly regulated
and are maintained at very low concentrations (reviewed in Ref.
61). Basal levels of free fatty acids in control L cells
(Fig. 4) comprised only a small
proportion (i.e., <5%) of total basal lipids (Fig. 3). In contrast,
supplementation of control L cells with palmitic acid-BSA or phytanic
acid-BSA increased the cellular levels of free fatty acids by 3.3-fold
(Fig. 4) and 3-fold (Fig. 4), respectively, compared with basal levels
(Fig. 4).
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Expression of intracellular fatty acid binding proteins differentially alters cellular levels of free (unesterified) fatty acids. Because the intracellular fatty acid binding proteins (SCP-2, SCP-x, and L-FABP) all interact with fatty acids, the possibility that expression of these proteins may increase the free fatty acid pool size was examined (Fig. 4). With one exception (i.e., SCP-2 overexpression and palmitic acid-BSA supplementation), expression of SCP-2, SCP-x, or L-FABP did not significantly increase the cellular level of free fatty acids under basal (BSA only), palmitic acid-BSA-, or phytanic acid-BSA-supplemented conditions (Fig. 4). On the contrary, expression of SCP-2, SCP-x, or L-FABP either had no effect or decreased the cellular level of free fatty acids (Fig. 4). Expression of SCP-x or L-FABP significantly (5.3- to 12-fold) decreased free fatty acid levels under basal (BSA only) or phytanic acid-BSA-supplemented conditions but not in palmitic acid-BSA-supplemented cells.
In summary, the expression of intracellular fatty acid binding proteins differentially decreased cellular levels of free fatty acids under basal conditions and in phytanic acid-BSA (but not palmitic acid-BSA)-supplemented cells in the following order: SCP-xSupplementation of L cells with either branched- or straight-chain
saturated fatty acids increased molar ratio of cholesterol to
phospholipid.
Culturing control L cells in the presence of palmitic acid-BSA or
phytanic acid-BSA resulted in 1.6- and 2-fold (P < 0.05) increased molar ratio of cholesterol to phospholipid,
respectively, compared with control cells fed BSA only (Fig.
5A). This effect was not due
to altered cellular phospholipid mass (Fig. 5B). Instead, supplementation with palmitic acid-BSA or phytanic acid-BSA
increased the cellular cholesterol mass 1.9- and 2.0-fold
(P < 0.05, n = 4-5; Fig.
5C), respectively, compared with control cells supplemented with BSA (no fatty acid) only.
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Intracellular fatty acid binding protein expression differentially alters saturated fatty acid-induced increase in cholesterol-to-phospholipid ratio. Under basal conditions, control L cells exhibited a cholesterol-to-phospholipid molar ratio near 0.4 (Fig. 5A). In addition, under basal conditions this ratio was not significantly affected by expression of intracellular fatty acid binding proteins because neither the cholesterol nor the phospholipid content was significantly altered (Fig. 5, B and C). Likewise, expression of intracellular fatty acid binding proteins did not prevent the palmitic acid-BSA-induced increase in molar ratio of cholesterol to phospholipid compared with control cells fed BSA only (Fig. 5A). In contrast, expression of intracellular fatty acid binding proteins significantly prevented the phytanic acid-BSA-induced increase in molar ratio of cholesterol to phospholipid compared with control cells fed BSA only (Fig. 5A). In fact, the molar ratio of cholesterol to phospholipid in phytanic acid-BSA-supplemented cells expressing SCP-2, SCP-x, or L-FABP (Fig. 5A) did not significantly differ from the corresponding cells in the basal state (Fig. 5A). Expression of intracellular fatty acid binding proteins protected the cell from phytanic acid-BSA-induced elevation in cholesterol-to-phospholipid ratio primarily by preventing cholesterol accumulation (Fig. 5C) and, in the case of SCP-x, also by lowering cellular phospholipid content (Fig. 5B).
In summary, expression of intracellular fatty acid binding proteins differentially protected L cells from phytanic acid-BSA-, but not palmitic acid-BSA-, induced increase in cellular cholesterol content and cholesterol-to-phospholipid ratio.Effect of methyl branching on saturated fatty acid-induced accumulation of neutral storage lipids of L cells: triacylglycerols and cholesteryl esters. Because saturated fatty acid supplementation increased the cellular cholesterol content (Fig. 5C), it was important to determine whether this increase was associated with increased formation of intracellular neutral storage lipids such as triacylglycerols and cholesteryl esters. Triacylglycerols and cholesteryl esters represent a major storage depot for fatty acids in control L cells (56).
Under basal conditions the triacylglycerols (Fig. 6A) comprised only ~5% of total lipids (Fig. 3). In contrast, supplementing the cells with palmitic acid-BSA (Fig. 6A) increased the triacylglycerol mass by 7.6-fold (P < 0.001) compared with control cells supplemented with BSA only (Fig. 6A). Control L cells supplemented with phytanic acid-BSA exhibited a 6.5-fold (P < 0.001) increased triacylglycerol mass (Fig. 6A) compared with control cells fed BSA only (Fig. 6A). Thus both branched- and straight-chain saturated fatty acids similarly induced ~7-fold the accumulation of neutral triacylglycerols. Cholesteryl esters under basal conditions (Fig. 6B) accounted for ~25% of total lipid in control L cells (Fig. 3). Palmitic acid-BSA supplementation induced a 1.6-fold increase in cellular cholesteryl ester accumulation (Fig. 6B), much less than observed for triacylglyceride accumulation (Fig. 6A). In contrast, phytanic acid-BSA supplementation did not significantly increase the cellular cholesteryl ester mass (Fig. 6B).
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Effect of intracellular fatty acid binding protein expression on saturated fatty acid-induced accumulation of neutral storage lipids: triacylglycerols and cholesteryl esters. The expression of intracellular fatty acid binding proteins did not exhibit a consistent pattern of uniformly reducing the saturated fatty acid-induced accumulation of triacylglycerols and cholesteryl esters (Fig. 6). Overall, expression of the intracellular fatty acid binding proteins (SCP-2, SCP-x, and L-FABP) did not prevent formation of triacylglycerols for storage compared with control cells supplemented with the same fatty acids (Fig. 6A). Only in the case of SCP-x expression was a partial reduction in triglyceride accumulation observed on palmitic acid-BSA (but not BSA-phytanic acid) supplementation. In general, fatty acid binding protein expression appeared more effective in reducing the accumulation of cholesteryl esters (Fig. 6B) than of triacylglycerols (Fig. 6A) induced by palmitic acid-BSA or phytanic-BSA supplementation.
Roles of fatty acid methyl branching and intracellular fatty acid
binding protein expression in saturated fatty acid uptake in L cells.
Together, the data in the preceding sections suggested that SCP-2,
SCP-x, and L-FABP expression differentially modulated the phytanic
acid- vs. palmitic acid-induced lipid accumulation in part by
decreasing uptake of phytanic, but not palmitic, acid. To determine
whether this is the case, total uptake of [3H]palmitic
and [3H]phytanic acid was examined in control and SCP-2-,
SCP-x-, and L-FABP-expressing cells. In control cells, the uptake of
straight-chain saturated fatty acid (i.e., [3H]palmitic
acid) was essentially the same as that of the methyl-branched chain
saturated fatty acid (i.e., [3H]phytanic acid) (Fig.
7). However, expression of intracellular fatty acid binding proteins differentially affected
[3H]palmitic and [3H]phytanic acid uptake
(Fig. 7). None of the fatty acid binding proteins inhibited
[3H]palmitic acid; in fact, expression of L-FABP
stimulated [3H]palmitic acid uptake by ~30%
(P < 0.05). In contrast, expression of SCP-2, SCP-x,
and L-FABP significantly (P < 0.05) inhibited the
uptake of [3H]phytanic acid by 2.8-, 7.5-, and 2.3-fold,
respectively, compared with control cells (Fig. 7). In summary, only
L-FABP expression enhanced uptake of [3H]palmitic acid,
whereas the expression of all three intracellular fatty acid binding
proteins (SCP-2, SCP-x, and L-FABP) inhibited uptake of
[3H]phytanic acid. Thus reduced
[3H]phytanic acid uptake (Fig. 7) was consistent with the
reduced total lipid accumulation exhibited in SCP-2-, SCP-x-, and
L-FABP-expressing cells supplemented with phytanic acid-BSA (Fig. 3).
Furthermore, the lack of inhibition of [3H]palmitic acid
uptake (Fig. 7) in the fatty acid binding protein-expressing cells was
consistent in general with the lesser ability of intracellular fatty
acid binding protein-expressing cells to prevent the palmitic acid-induced lipid accumulation (Fig. 3).
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Differential oxidation of branched- vs. straight-chain saturated fatty acids: effects of intracellular binding protein expression. As indicated in the introductory paragraphs of this article, both in vitro studies and some animal studies suggested that intracellular fatty acid binding proteins (SCP-2, SCP-x, L-FABP) may function in the oxidation of straight-chain fatty acids and, at least in the case of SCP-x, in peroxisomal oxidation of branched-chain fatty acids (2, 80). However, neither the role of SCP-x in straight-chain (palmitate) oxidation nor the role of SCP-2 and L-FABP in either type of fatty acid oxidation has been directly addressed in any cell type or animal. The differential protective effects of intracellular fatty acid binding proteins on phytanic, but not palmitic, acid-induced lipid accumulation noted in the preceding sections may, at least in part, be due to these fatty acid binding proteins differentially facilitating the oxidation of phytanic acid compared with that of palmitic acid.
Significant differences were observed between the total oxidation of straight- and branched-chain fatty acids in control L cells. The total oxidation of [3H]palmitic acid was nearly fivefold (P < 0.001) greater than that of [3H]phytanic acid (Table 2). Although expression of intracellular fatty acid binding proteins did not significantly affect the total oxidation of [3H]palmitic acid, total oxidation of [3H]phytanic acid was 3.8-, 9.3-, and >100-fold slower (P < 0.01) in SCP-2-, SCP-x-, and L-FABP-expressing cells, respectively, compared with total [3H]phytanic acid oxidation in control cells (Table 2).
|
Effect of SCP-2, SCP-x, and L-FABP expression on incorporation of
radiolabeled palmitic acid and phytanic acid into esterified lipids.
Because only a small proportion of [3H]palmitic acid and
[3H]phytanic acid taken up was oxidized in the control
cells, i.e., 21% and 5%, respectively (Table 2), it was important to
determine whether the remainder was esterified or remained as free
fatty acids in the cells. Only 14% of [3H]palmitic acid
taken up was esterified in control L cells supplemented with palmitic
acid-BSA (Fig. 8). Similarly, only a
small portion (i.e., 8.6%) of [3H]phytanic acid taken up
was esterified in control L cells supplemented with phytanic acid-BSA
(Fig. 8). However, this was 39% less (P < 0.05) than
observed with [3H]palmitic acid that was taken up.
Expression of SCP-x and L-FABP (but not SCP-2) increased the portion of
[3H]palmitic acid taken up that was esterified 1.6- and 1.3-fold, respectively (Fig. 8). In contrast, expression of SCP-2,
SCP-x, and L-FABP decreased the percentage of
[3H]phytanic acid taken up that was esterified by 2.4-, 5.4-, and 1.8-fold, respectively (Fig. 8).
|
Effect of SCP-2, SCP-x, and L-FABP expression on palmitic acid-BSA-
and phytanic acid-BSA-induced cytotoxicity in L cell fibroblasts.
The above differential effects of intracellular fatty acid binding
proteins on palmitic acid- vs. phytanic acid-induced lipid accumulation
as well as uptake and oxidation of the respective fatty acids suggested
that SCP-2, SCP-x, or L-FABP might also alter the toxicity of these
fatty acids, especially that of phytanic acid. In all of the preceding
studies on palmitic acid-BSA and phytanic acid-BSA effects (i.e., lipid
accumulation, uptake, oxidation, esterification) the cells were
incubated with complexes made of 25 µM fatty acid and ~90 µM BSA
for a period of 6 h. Under these conditions, no overt cytotoxicity
(based on protein/dish, cell number/dish, live/dead) was noted for any
of the cell lines examined (data not shown). However, at longer
incubation times (12 h) and/or higher concentrations of saturated
fatty acids significant cytotoxicity was observed.
|
|
|
Compensatory changes in levels of other fatty acid
binding/transport proteins: effect of SCP-2, SCP-x, or L-FABP and
effect of saturated fatty acids.
Although no alterations in plasma membrane fatty acid transport
proteins were detected in SCP-x/SCP-2 gene-ablated mice, compensatory upregulation of L-FABP was observed (81). Therefore, the
possibility that SCP-2, SCP-x, or L-FABP expression in transfected L
cells could elicit altered expression of other proteins involved in lipid uptake and transport was considered. The expression levels of the
cytosolic fatty ACBP as well as SCP-2, SCP-x, and L-FABP were
determined by Western blotting. Expression levels of ACBP were
unchanged in all cell lines (Fig. 11).
SCP-2 in the L-FABP-expressing cells and L-FABP in the SCP-2- and
SCP-x-expressing cells were also unchanged (data not shown).
|
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DISCUSSION |
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Almost nothing is known regarding potential effects of intracellular fatty acid binding proteins on lipid accumulation and toxicity of high levels of branched- and straight-chain fatty acids. To investigate these possibilities in detail, three intracellular fatty acid binding proteins (SCP-2, SCP-x, L-FABP) were individually overexpressed in transfected L cells. L cell fibroblasts were an appropriate model because protein levels of L-FABP, SCP-2, SCP-x (Fig. 1), or other known members of the intracellular fatty acid binding protein family were below the level of detection (37). The three transfected L cell lines overexpressed SCP-x, SCP-2, and L-FABP at levels (0.01%, 0.036%, and 0.40% of cytosol protein, respectively) and proportions (10- to 40-fold more L-FABP than SCP-2 or SCP-x) found in animal tissues (reviewed in Refs. 23, 37, 47, 71). The transfected L cells overexpressing 58-kDa SCP-x partially posttranslationally processed the 58-kDa SCP-x to yield detectable 43-kDa thiolase and 13-kDa SCP-2 (~2-fold increased), consistent with earlier data (4). Examination of a variety of transfected clones expressing different levels of intracellular fatty acid binding proteins showed that the direction of their effect on lipid metabolism was similar and dose responsive (4, 37, 50, 55). The highest-expression clone for each intracellular fatty acid binding protein with levels closest to physiological significance was used for the studies presented here. This allowed analysis of the effect of the individually expressed proteins on lipid accumulation and toxicity induced by high levels of branched- and straight-chain fatty acids, independent of the presence and/or concomitant upregulation of other fatty acid binding proteins. It should be noted that L cells contain the appropriate machinery for fatty acid uptake and esterification (reviewed in Ref. 47) and fatty acid oxidation (e.g., mitochondria and peroxisomes; Fig. 2, Refs. 24, 68, 72, 78) and have been used to examine fatty acid-induced lipid accumulation and toxicity (25, 26). Fibroblasts have been used by many laboratories to study human defects of fatty acid oxidation (reviewed in Refs. 93, 98). The data presented here contributed significantly to our understanding of the roles of intracellular fatty acid binding proteins on branched- vs. straight-chain saturated fatty acid-induced cellular lipid accumulation, uptake, oxidation, and cytotoxicity.
First, it was shown that the methyl-branched saturated fatty acid (phytanic acid) was much more cytotoxic than the straight-chain saturated fatty acid (palmitic acid), even when L cells were exposed to much lower concentration and/or exposure time with phytanic acid than palmitic acid. Interestingly, the greater cytotoxicity of phytanic acid in L cells did not correlate with any differences in total lipid accumulation or specific lipid class accumulation. Similar lack of correlation of lipid accumulation with cytotoxicity has also been reported for saturated vs. unsaturated fatty acids in L cells in that both similarly induce lipid accumulation but only the saturated fatty acids are cytotoxic (25). Because the greater cytotoxicity of phytanic acid was not due to any differences in uptake of [2,3-3H]phytanic acid vs. [9,10-3H]palmitic acid, two factors appeared to be consistent with the greater cytotoxicity of phytanic acid. 1) [2,3-3H]phytanic acid oxidation was more than fourfold slower than that of [9,10-3H]palmitic acid. 2) [2,3-3H]phytanic acid esterification was nearly 40% slower than [9,10-3H]palmitic acid. The lower oxidation and esterification of phytanic vs. palmitic acid may be accounted for by the much lower activity of fatty acyl CoA synthases with branched- vs. straight-chain fatty acids (99). Together with the fact that the total mass of unesterified free fatty acid in the palmitic acid- and phytanic acid-supplemented cells did not differ in control cells (Fig. 4), these data suggest that a greater proportion of phytanic acid remained unesterified in the free fatty acid fraction, where it was apparently more cytotoxic than the unesterified palmitic acid. Interestingly, high levels of saturated fatty acids inhibit cell growth and cause cell death by disruption of cellular metabolism because of excessive lipid accumulation of triglycerides and free fatty acids to the endoplasmic reticulum (ER), where they crystallize (25). In contrast, high levels of unsaturated fatty acids typically accumulate in cytoplasmic lipid droplets, a storage depot (25). The incorporation of branched-chain saturated fatty acids into membranes induces the formation of abnormal, inverted nonbilayer phases (79) and increases membrane fluidity (17, 21, 43). Both factors may contribute significantly to altered membrane functions (43). Although their uptake was similar under the conditions examined, the fourfold lower oxidation and 40% lower esterification of phytanic acid vs. palmitic acid would suggest that higher levels of branched-chain fatty acids accumulated in L cell membranes and may therefore be more damaging to the membranes and their functions.
Second, under conditions in which fatty acid-induced lipid accumulation
was observed, the expression of the intracellular fatty acid binding
proteins did not protect L cell fibroblasts from the cytotoxicity of
saturated branched-chain phytanic acid. Although expression of these
proteins (SCP-2, SCP-x, and L-FABP) reduced the phytanic acid-induced
lipid accumulation, these proteins concomitantly enhanced the
cytotoxicity of phytanic acid. The enhanced cytotoxicity was not due to
increased [2,3-3H]phytanic acid uptake or to increased
levels of fatty acid transporters in the plasma membrane. Instead,
increased toxicity correlated with significant inhibition of
[2,3-3H]phytanic acid oxidation (in the order L-FABP
SCP-x
SCP-2) and inhibition of [2,3-3H]phytanic acid
esterification (in the order SCP-x > SCP-2, L-FABP). These
effects of intracellular fatty acid binding protein expression on
phytanic acid were in contrast to those observed with palmitic acid:
unaltered (or slightly increased) [9,10-3H]palmitic acid
uptake, unaltered (or only modestly decreased) [9,10-3H]palmitic acid oxidation, and unaltered (or
increased) [9,10-3H]palmitic acid esterification. Thus
expression of intracellular fatty acid binding protein increased the
proportion of [2,3-3H]phytanic acid present in
unesterified form more than that of [9,10-3H]palmitic
acid in unesterified form. Increased cytotoxicity with cells
overexpressing these proteins would be expected if the unesterified branched-chain fatty acids accumulated in membranes, leading to the
complications described above.
Third, the present studies for the first time directly examined the effect of intracellular fatty acid binding protein expression in intact cells on fatty acid oxidation in intact cells. At levels (i.e., 25 µM) at which both these saturated fatty acids induce intracellular lipid accumulation, expression of all three fatty acid binding proteins inhibited total phytanic, but not palmitic, acid oxidation by 35-99%. Even when expressed as a percentage of fatty acid taken up and oxidized, expression of all three proteins much more strongly inhibited oxidation of phytanic acid. In contrast, at low concentrations (i.e., 50 nM phytanic or palmitic acid) expression of fatty acid binding protein did not inhibit phytanic or palmitic acid oxidation (data not shown). Thus, in contrast to studies with stimulatory effects of fatty acid binding proteins on palmitic acid oxidation by isolated mitochondria in vitro (62), expression of these proteins in L cells did not enhance palmitic or phytanic acid oxidation in intact cells. These fatty acid binding proteins did not inhibit phytanic acid oxidation by decreasing phytanic acid uptake or the levels of plasma membrane transport proteins (Fig. 10) or other intracellular fatty acid/fatty acyl CoA binding proteins (Fig. 10). Furthermore, the inhibition was specific for the branched-chain compared with the straight-chain saturated fatty acid. The molecular mechanism accounting for this specificity is not yet known. Future studies are needed.
Fourth, the observation that the L-FABP expression elicited the highest cytotoxicity of phytanic acid in L cells may help explain recent observations of phytanic acid toxicity in SCP-x/SCP-2 gene-ablated mice (81). In that study, it was observed that phytanic acid toxicity was dramatically increased after feeding a diet high in phytanic acid to SCP-x/SCP-2 gene-ablated mice but not control mice (81). The latter finding, at first glance, apparently contradicted the observations presented here with control L cells (no SCP-x or SCP-2) vs. transfected L cells overexpressing SCP-x or SCP-2. However, this apparent discrepancy may be readily explained by the concomitant fourfold upregulation of L-FABP in livers of the SCP-x/SCP-2 gene-ablated mice. L-FABP upregulation to near millimolar levels was much greater than the loss of micromolar amounts of SCP-x/SCP-2 in the knockout mice and could therefore account for the increased phytanic acid toxicity, consistent with the enhanced cytotoxicity of phytanic acid in L-FABP-overexpressing L cells reported here.
Several additional factors contribute to the enhanced phytanic acid cytotoxicity in cells overexpressing intracellular fatty acid binding proteins. 1) Expression of intracellular fatty acid binding proteins decreased the ratio of cholesterol to phospholipid in the presence of phytanic, but not palmitic, acid. Decreasing the plasma membrane cholesterol-to-phospholipid ratio significantly increases membrane fluidity and inhibits Na+-K+-ATPase activity (34). Agents that fluidize plasma membranes significantly inhibit Na+-K+-ATPase activity (89, 90). Fatty acid-induced cytotoxicity is observed in L cells when membrane fluidity is outside an optimal range (75). 2) Expression of intracellular fatty acid binding proteins increased the levels of unesterified, free phytanic acid much more than free palmitic acid. Branched-chain fatty acids are much more membrane disruptive than straight-chain saturated fatty acids (17, 21, 25, 26, 43, 48). 3) The preferential accumulation of free branched-chain fatty acids in intracellular fatty acid binding protein-expressing cells can differentially alter intracellular signaling pathways. Branched-chain saturated fatty acids alter intracellular signaling pathways (16), but in a manner different from the effect of straight-chain saturated fatty acids (reviewed in Ref. 13). 4) The increase in free phytanic acid compared with free palmitic acid induced by intracellular fatty acid binding protein expression can differentially affect nuclear regulatory proteins. In contrast to straight-chain saturated fatty acid, phytanic acid is a potent peroxisome proliferator-activated receptor (PPAR) ligand and transcriptional activator (102). SCP-2 expression was increased by phytanic acid-BSA, but not palmitic acid-BSA, supplementation in all the cell lines examined (data not shown). Interestingly, an imperfect DR1 element, which is potentially a binding site for PPAR, was reported in the promoter region of SCP-2, making this a distinct possibility (58). These data suggest the possibility that phytanic acid could inappropriately affect PPAR transcriptional activity to upregulate additional fatty acid binding proteins that enhance phytanic acid transport to peroxisomes. Consistent with this possibility, phytanic acid induced L-FABP expression fourfold in SCP-x/SCP-2 gene-ablated mice (81).
In summary, the studies presented here for the first time directly examined whether expression of intracellular fatty acid binding proteins (SCP-2, SCP-x, L-FABP) could protect cells from lipid accumulation and/or cytotoxicity induced by high levels of branched- or straight-chain saturated fatty acids. It was shown that, although SCP-x expression partially protected the cells against straight-chain saturated fatty acid (palmitic acid)-induced lipid accumulation, neither SCP-2 nor L-FABP did so. In contrast, although expression of all three proteins (SCP-2, SCP-x, and L-FABP) partially protected the cells against branched-chain saturated fatty acid (phytanic acid)-induced lipid accumulation, all three intracellular binding proteins enhanced the cytotoxicity of phytanic acid but not palmitic acid. The enhancement of phytanic acid cytotoxicity appeared to be due primarily to inhibition of phytanic acid oxidation and esterification rather than alteration of its uptake via plasma membrane transporters.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants DK-41402 and GM-31651.
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FOOTNOTES |
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Address for reprint requests and other correspondence: F. Schroeder, Dept. of Physiology and Pharmacology, Texas A&M Univ., TVMC, College Station, TX 77843-4466 (E-mail: Fschroeder{at}cvm.tamu.edu).
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. Section 1734 solely to indicate this fact.
April 24, 2002;10.1152/ajpcell.00586.2001
Received 10 December 2001; accepted in final form 18 April 2002.
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