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Articles

The F508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Alters Control of Essential Fatty Acid Utilization in Epithelial Cells¹

Farah N. Bhura-Bandali^*, Miyoung Suh^*, S. F. Paul Man and M. Thomas Clandinin^*,2

^* Nutrition and Metabolism Research Group, Department of Agricultural, Food and Nutritional Science and Department of Medicine, University of Alberta, Edmonton, AB, Canada T6G 2P5

²To whom correspondence should be addressed.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Essential fatty acid (EFA) incorporation into phospholipid is influenced by chloride channels, suggesting that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) may regulate aspects of EFA metabolism. The objective of this study was to determine whether the F508 mutation in the CFTR lowers 18:2(n-6) levels in phospholipid. Control cells, CF cells and CF cells transfected with the "normal" CFTR gene or the F508 CFTR gene were cultured for 3–5 d and used to determine [1-¹⁴C]18:2(n-6) incorporation into cell lipids. CF cells exhibited low 18:2(n-6) levels in phospholipid, reduced [1-¹⁴C]18:2(n-6) incorporation into phospholipid (50% of control) and greater [1-¹⁴C]18:2(n-6) incorporation into the triacylglycerol fraction (400% of control; P < 0.05). Kinetic modeling of time course data for [1-¹⁴C]18:2(n-6) incorporation revealed a loss of metabolic control over the intracellular partitioning of 18:2(n-6) between phospholipid and triacylglycerol pools in CF cells. Expression of the normal CFTR gene in transfected CF cells increased chloride efflux and the incorporation of [1-¹⁴C]18:2(n-6) into phospholipid and triacylglycerol fractions. The increased incorporation of [1-¹⁴C]18:2(n-6) into phospholipid was attributed to significantly increased incorporation of [1-¹⁴C]18:2(n-6) into phosphatidylcholine and phosphatidylinositol. In CF cells expressing the defective F508 CFTR gene, conversion of [1-¹⁴C]18:2(n-6) to 20:4(n-6) by desaturation-chain elongation was 1.8-fold greater (P < 0.05) than observed for CF cells transfected with the normal gene. The observations suggest that CF results in a defect in the utilization of 18:2(n-6), which is attributed in part to the defective CFTR.

KEY WORDS: • cystic fibrosis • essential fatty acids • humans • epithelial cell • membrane

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Numerous essential cellular functions depend on biological membranes (Yeagle 1989 ). Proteins and lipids exhibit both independence and synergy in the maintenance of membrane structure and function. Defects in fundamental membrane properties can have profound implications for the usual physiologic milieu of the cell. Cystic fibrosis (CF),³ the most common autosomal recessive fatal disease in Caucasians (Smith 1995 ), exemplifies defects in both membrane structure and function. At the cellular level, the disease is characterized by abnormalities in water and electrolyte transport. Clinically, CF leads to chronic pulmonary infection, pancreatic insufficiency and abnormal levels of electrolytes in the sweat (Quinton 1990 , Smith 1995 , Tizzano and Buchwald 1992 ). The gene responsible for CF encodes a membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), which is a cAMP-dependent chloride channel (Anderson et al. 1991 , Bear et al. 1992 , Drumm et al. 1990 ) with other regulatory functions (Bradbury et al. 1992 , Hyde et al. 1990 ), contributing to the pleiotropic nature of the disease. Genetic analysis has revealed >700 mutations in the CFTR; the deletion of a phenylalanine residue at position 508 (F508) is the most common mutation, comprising nearly 70% of CF patients (Chakravarthy et al. 1986 , Leidke 1992 , Tizzano et al. 1993 ). A large majority of patients homozygous for F508 exhibit pancreatic insufficiency with heightened disease severity.

For decades, plasma and tissue lipids of patients with CF have been shown to exhibit low essential fatty acid (EFA) (linoleic acid) levels (Clandinin et al. 1995 , Farrell et al. 1985 , Lloyd-Still et al. 1981 , Tizzano et al. 1994 ), believed to reflect inadequate intake. Reduced linoleic acid [18:2(n-6)] levels, despite pancreatic sufficiency and adequate dietary supplementation, have been reported (Clandinin et al. 1995 , Lloyd-Still et al. 1981 , Parsons et al. 1988 ), which argues against a nutritional cause. Despite low linoleic acid levels, normal to increased levels of arachidonic [20:4(n-6)] occur in plasma and tissue lipids of CF patients, and the production of eicosanoids is elevated (Carlstedt-Duke et al. 1986 , Hubbard and Dunn 1980 , Lloyd-Still et al. 1981 , Stead et al. 1986 , Strandvik 1996 ). Thus, it appears that the characteristic EFA profile of CF patients is unlike the "classic" EFA deficiency but is suggestive of a defective regulation of EFA metabolism. Active incorporation of fatty acids is a dynamic process defining the fatty acids found in cell lipids (Clandinin 1976 ) and is influenced by chloride channels (Kang et al. 1992 ). Inhibition of both CFTR and non-CFTR chloride channels reduces EFA incorporation into phospholipids (Kang et al. 1992 ), suggesting a relationship between chloride transport and EFA metabolism. The objective of this study was to examine whether the F508 mutation in the CFTR reduces the level of 18:2(n-6) in membrane lipids of cultured CF pancreatic epithelial cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Radioactive materials [1-¹⁴C]18:2(n-6) (3 Bq/mmol) and [³⁶Cl^-] NaCl in aqueous solution (33.26 Bq/mmol chlorine) of >90% purity were purchased from NEN Research Products (DuPont Canada, Mississauga, Canada) and used without further purification. Unlabeled 18:2(n-6), lipid standards and other biochemicals were obtained from Sigma Chemical (St. Louis, MO) and Gibco BRL (Gaithersburg, MD). All organic solvents were redistilled before use.

Cell lines and maintenance.

Cystic fibrosis and control human pancreatic epithelial cell lines of ductal origin, CF cells (CFPAC-1) and "normal" (control) cells (PANC-1) (Lieber et al. 1975 ), were purchased from ATCC (Rockville, MD). CF cells stably transfected with the "normal" CFTR gene or the defective F508 CFTR gene (Drumm et al. 1990 ) were generously provided by Dr. R. A. Frizzel (Pittsburgh, PA).

Cell lines were suspended in Dulbecco’s modified Eagle’s medium, supplemented with 5 or 10% (v/v) fetal bovine serum (FBS) and antibiotic-antimycotic solution (Gibco BRL), containing 10,000 U penicillin G, 10 mg streptomycin sulfate and 25 mg/L amphotericin B. Cells were plated on 25-cm² tissue culture plates and cultured at 37°C in 5% CO₂ at 98% relative humidity in a tissue culture incubator (Jouan CR4.11; Forma Scientific, Marietta, OH). Culture medium was changed every 2–3 d until confluent monolayers were formed for use in biochemical assays.

[1-¹⁴C]18:2(n-6) incorporation.

Confluent monolayers of cells were washed with Hank’s balanced salt solution (HBSS; Gibco BRL) and detached from the flask surface with 2.5 g/L trypsin-EDTA (1 mmol/L EDTA, 400 g/L NaCl) solution. The cell suspension was neutralized with culture medium containing FBS, collected in a centrifuge tube and centrifuged at 157 x g at 25°C for 3 min. Cells were then washed once with medium without FBS and an aliquot removed for cell counts and viability determinations. Using a hemocytometer, the average number of cells was counted (2.0–3.0 x 10⁶) and viability per experiment estimated (85–90%). Cells were replated onto 60-mm (fatty acid incorporation studies) or 100-mm (fatty acid desaturation studies) diameter tissue culture plates in 2 mL medium without FBS. A mixture of labeled and unlabeled fatty acids suspended by sonication in sterile 50 g/L bovine serum albumin was added in a 100 µL volume to give a final fatty acid concentration of 50 µmol/L and 132 MBq of [1-¹⁴C]18:2(n-6). Cells were incubated for various time points (0–4 h) under conditions described above. After incubation, cells were harvested using a rubber policeman and plates were washed twice with HBSS. Culture medium and washes were removed by centrifugation and cell pellets were further washed twice with HBSS. Cell pellets were suspended in 1 mL HBSS and then used for lipid extraction and analyses.

Lipid extraction and analysis.

Cellular lipid was extracted with 20 mL chloroform/methanol (2:1, v/v; Folch et al. 1957 ) containing 0.005% (v/v) ethoxyquin as antioxidant. After vigorous mixing, 0.5 g/L calcium chloride was added, and the tube contents were mixed and refrigerated overnight at 4°C. The chloroform phase containing lipid was collected, dried under a gentle stream of nitrogen and stored in sealed tubes at -70°C until further analysis.

Lipid classes were separated using TLC. TLC plates were cleaned with hexane and activated at 110°C for 60 min. Total phospholipid and triacylglycerol was separated using 250-µm Silica Gel G plates (20 x 20 cm, Fisher Scientific, Ottawa, Canada) in a solvent system comprising petroleum either/diethyl ether/acetic acid (160:40:2, v/v/v) for 25–30 min (Skipski and Barday 1969 ). Individual phospholipids [phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE)] were separated on 250-µm Silica Gel H plates (20 x 20 cm) in a solvent system of chloroform/methanol/2-propanol/triethylamine/2.5 g/L potassium chloride (60:18:50:36:12, by volume) for 90 min (Touchstone et al. 1980 ). Fatty acids were methylated using 140 g/L boron triflouride/methanol reagent in hexane at 110°C for 60 min (Hargreaves and Clandinin 1987 ). Triacylglycerol was saponified before methylation with 0.5 mol/L methanolic potassium hydroxide and allowed to reflux at 110°C for 60 min.

Argentation TLC was used to resolve fatty acid methyl esters (FAME) on the basis of the degree of unsaturation (Suh et al. 1994 ). [1-¹⁴C]18:2(n-6) labeled samples were applied on TLC plates impregnated with AgNO₃. Silica Gel H plates (250 µm; 20 x 20 cm) were developed in a 100 g/L AgNO₃ solution in water in a TLC tank for 60 min. The plates were dried for 3 min in the dark and activated in a 110°C oven for 1 h. Each sample and standards were spotted on the plate in a narrow band and the plates were developed twice in a solvent system of hexane/diethyl either/acetic acid/toluene/acetone (50:4:2:40:4, by volume) for 1 h and then for 30 min. Plates were dried at room temperature for 3 min and standards visualized with 1 g/L 2'7'-dichlorofluorescein in 95% (wt/v) ethanol. Bands of fatty acids, from saturated to those containing up to six double bonds, were separated using this method. 22:4(n-6) could be present in the band of 20:4(n-6); however, the level of 22:4(n-6) produced is very small and would be essentially undetectable. A high level of 22:4(n-6) in the 20:4(n-6) band would not change the conclusions of the study.

Analysis of FAME was carried out by a fully automated Varian Vista 6000 gas-liquid chromatograph (Georgetown, Canada) equipped with a flame-ionization detector and a Varian Vista 654 data system (Hargreaves and Clandinin 1987 ). The analytical conditions used separated all saturated, mono- and polyunsaturated fatty acids ranging from C-14 to C-24.

Liquid scintillation counting.

[1-¹⁴C]18:2(n-6) labeled samples separated by TLC were scraped directly into 20-mL plastic scintillation vials and 10 mL scintillation cocktail (ScintiSafe Econo 1, Fisher Scientific) was added. Radioactivity was counted in a Beckman LS 5801 Liquid Scintillation Counter (Irvine, CA) with a counting efficiency of 94–95%. Quench was monitored by the "H-number" method and counts were corrected for differences in counting efficiency.

Chloride efflux.

Efflux of radiolabeled chloride was used to assess chloride conductance of cell lines (Dunn et al. 1994 ). Cells grown to confluence on 35-mm culture dishes were washed with bicarbonate-free Krebs-Ringer solution, loaded with 162 MBq of [³⁶Cl-] NaCl(aq) and incubated for 2 h at 37°C in 5% CO₂ at 98% relative humidity in a tissue culture incubator. After incubation, the monolayer was washed four times and isotope efflux was measured by replacing 1 mL Krebs-Ringer solution at 15-s intervals. After eight baseline measurements, 10 µmol/L forskolin and 100 µmol/L 3-isobutyl-1-methylxanthine were added and an additional seven stimulated measurements were taken. Chloride efflux was calculated as [³⁶Cl-] released from the monolayer over time and expressed as the ratio of the released (stimulated)/ released (baseline).

Statistical analysis.

Difference between control and CF cells or CF cells transfected with the "normal" CFTR gene or F508 CFTR gene was determined using Student’s t test with P < 0.05 considered significant. Data from time course experiments were also analyzed using the SAAM II Biological Modeling Program (Seattle, WA), which was designed to illustrate the movement of tracer material between different biological compartments. The program utilizes simple differential equations that are solved and fitted using mathematical and statistical functions (least-squares fitting). SAAM II determined the rate constants for the movement of [1-¹⁴C]18:2(n-6) between phospholipid and triacylglycerol fractions of control and CF cells. Significant difference between the rate constants was also determined using Student’s t test. Values are means ± SEM.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Fatty acid composition of control and cystic fibrosis cells.

The characteristic EFA profiles of phospholipids from CF and control cells indicated that these two cell types maintain different membrane 18:2(n-6) levels (Table 1 ). 18:2(n-6) was lower in PC and PE, and greater in PS of CF cells. The 20:4(n-6) levels in CF cell PS, PI and PE were 91, 105 and 53% higher than in the control cells, respectively. These results are in agreement with those previously reported for CF cells (Christophe et al. 1992 , Farrell et al. 1985 , Levy et al. 1989 , Parsons et al. 1988 ).

Incorporation of EFA into lipids of CF cells exhibiting the F508 mutation was distinct from that in control cells. Incorporation of [1-¹⁴C]18:2(n-6) into total phospholipid was lower, whereas incorporation into triacylglycerol was greater in CF cells compared with control cells (Fig. 1 ). There was less incorporation of [1-¹⁴C]18:2(n-6) into PC (3.7 ± 1.20 vs. 9.2 ± 0.94%, P < 0.05) and PE (0.6 ± 0.24 vs. 1.2 ± 0.16%, P < 0.05) in CF cells than in control cells. Considering the level of substrate incorporation into individual phospholipids, these data suggest that reduced incorporation into total phospholipid is largely a result of reduced incorporation into PC.

View larger version (16K): [in this window] [in a new window]   Figure 1. [1-14C]18:2(n-6) incorporation into phospholipid and triacylglycerol of control (PANC-1) and cystic fibrosis (CF, CFPAC-1) cells. Cells were replated and incubated with [1-14C]18:2(n-6) for 4 h. Lipids were extracted and lipid classes separated by TLC. Radioactivity was counted by liquid scintillation spectrometry. Values are means ± SEM, n = 7. *P < 0.05.

At each time point studied, incorporation of [1-¹⁴C]18:2(n-6) was higher in triacylglycerol in CF cells (P < 0.001) than in control cells (Fig. 2A ). By 4 h, <10% of the substrate was incorporated, leaving >90% of the substrate still in the medium. Thus, the substrate concentration in the medium was not limiting (Fig. 2) . For control cells, little incorporation of [1-¹⁴C]18:2(n-6) into the neutral lipid occurred over time. Substrate incorporation into total phospholipid did not differ between the cell types until the end of the incubation period when incorporation was reduced (P < 0.05) in CF cells (Fig. 2B ). Rate constants for incorporation of [1-¹⁴C]18:2(n-6) into phospholipid and triacylglycerol of control cells differed (P < 0.01), whereas in CF cells, the rate constants did not differ (Fig. 3 ), suggesting a loss of metabolic control in the CF cells.

View larger version (17K): [in this window] [in a new window]   Figure 2. Time course for incorporation of [1-14C]18:2(n-6) over 4 h into triacylglycerol (A) and phospholipid (B) of control (PANC-1) and cystic fibrosis (CFPAC-1) cells. Cells were replated and incubated with [1-14C]18:2(n-6) for 0, 1, 2, 3 or 4 h. Lipids were extracted and lipid classes separated by TLC. Radioactivity was counted by liquid scintillation spectrometry. Values are means ± SEM, n = 5. *P < 0.001)(A) and *P < 0.05 (B).

View larger version (27K): [in this window] [in a new window]   Figure 3. Kinetic modeling of the rate constants for the incorporation of [1-14C]18:2(n-6) over 4 h into triacylglycerol (TAG) and phospholipid (PL) of control (PANC-1) and cystic fibrosis (CFPAC-1) cells. The rate constants were determined by analyzing the isotope incorporation data illustrated in Figure 2A and B using the SAAM II Biological Modeling Program (Seattle, WA). Values (k = h-1) are means ± SEM, n = 5. Difference between rate constants is indicated. *P < 0.01.

Before undertaking [1-¹⁴C]18:2(n-6) incorporation studies on transfected CF cells, functional studies of chloride conductance were performed. Chloride efflux was >200% higher for cells transfected with the "normal" gene compared with cells transfected with the defective CF (F508) gene (1.3 ± 0.13 vs. 0.4 ± 0.03, P < 0.001). Chloride efflux was also measured in control and CF cells (0.45 ± 0.03 vs. 0.50 ± 0.02, respectively) to enable comparison with the extent of the increase in chloride efflux resulting from transfection of the CF cell type with the "normal" CFTR gene. Transfecting CF cells with this gene increased chloride transport apparently to levels greater than characteristic of control cells.

Labeled substrate incorporation into CF cells transfected with the "normal" gene was higher for both total phospholipid (9.1 ± 0.92 vs. 6.0 ± 0.60%, P < 0.03) and triacylglycerol fractions (9.2 ± 1.27 vs. 4.8 ± 0.45%, P < 0.03) compared with cells transfected with the defective F508 gene. Increased [1-¹⁴C]18:2(n-6) incorporation into triacylglycerol was an unexpected finding because CF cells incorporated more [1-¹⁴C]18:2(n-6) into triacylglycerols compared with the control cells (Fig. 1) . Comparisons among CF cells transfected with the "normal" CFTR or defective CF cells for [1-¹⁴C]18:2(n-6) incorporation into triacylglycerol revealed that all three CF cell lines (CF cells transfected with "normal" gene, CF cells transfected with defective F508 gene and CF cells) incorporated significantly more substrate into the triglyceride fraction compared with control cells (9 ± 1.8, 4.3 ± 0.4 and 5.4 ± 1.7%, respectively, vs. 1.3 ± 0.1% in control cells).

Separation of phospholipids revealed greater [1-¹⁴C]18:2(n-6) incorporation (expressed as % of total label) into PC (7.3 ± 0.82 vs. 4.3 ± 0.33%), PA (0.2 ± 0.02 vs. 0.1 ± 0.01%) and PI (1.2 ± 0.14 vs. 0.8 ± 0.06%) in CF cells transfected with the "normal" gene compared with cells transfected with the defective F508 CF gene (P < 0.05).

Conversion of [1-¹⁴C]18:2(n-6) to its desaturation and elongation products: distribution of labeled fatty acid into total and individual phospholipids.

Desaturation and elongation of radiolabeled 18:2(n-6) were determined in control, CF and both types of transfected CF cells. These cell types can desaturate and elongate [1-¹⁴C]18:2(n-6), indicating the presence of active 6- and 5-desaturase enzyme systems. It is not known which esterified form of 18:2(n-6) is the actual substrate for desaturation. Compared with control cells, CF cells incorporated more [1-¹⁴C]18:2(n-6) into total phospholipid (Table 2 ), PC and PE (Table 3 ), even though the 18:2(n-6) pool was smaller in these phospholipids of CF cells, suggesting a higher rate of turnover of 18:2(n-6). Differences between distribution of desaturation-elongation products of [1-¹⁴C]18:2(n-6) in control and CF cells were observed for trienes in PE (Table 3) . Expression of the "normal" gene in CF cells resulted in more [1-¹⁴C]18:2(n-6) and less [1-¹⁴C]20:4(n-6) in the phospholipid fraction compared with CF cells transfected with the defective F508 CF gene (Table 4 ). Separation of phospholipids revealed greater incorporation of [1-¹⁴C]18:2(n-6) into both PC and PE of CF cells containing the "normal" gene compared with F508 transfected CF cells (Table 5 ).

View this table: [in this window] [in a new window]   Table 4. The incorporation of [1-14C]18:2(n-6) into desaturation-elongation products in phospholipids of "normal" and F508 CFTR-transfected cystic fibrosis (CF) cells12

View this table: [in this window] [in a new window]   Table 5. The incorporation of [1-14C]18:2(n-6) of desaturation-elongation products in phosphatidylcholine and phosphatidylethanolamine of "normal" and F508 CFTR-transfected cystic fibrosis (CF) cells12

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The high level of 18:2(n-6) incorporation into triacylglycerol in transfected and nontransfected CF cell lines implies that this effect may not require a functional CFTR. It is not known whether the increased incorporation of 18:2(n-6) into triacylglycerol is related to enhanced triacylglycerol synthesis to accommodate an influx of fatty acid or whether this pool of neutral lipid acts as a trap for acyl chains released from phospholipids (Chakravarthy et al. 1986 ). Biochemical connections between the defective CFTR and functional anomalies in lipid metabolism observed in CF cells remain unclear (Chakravarthy et al. 1986 , Leidke 1992 , Smith et al. 1995 , Tizzano et al. 1993 ). All aspects of the CF phenotype involving EFA utilization may not be resolved by expression of the "normal" CFTR in CF cells (Drumm et al. 1990 ). Recent evidence in CFTR knockout mice has suggested that feeding 22:6(n-3), an EFA, normalizes the disease-related changes that occur in epithelial cells and in intestinal mucosa (Freedman et al. 1999 ). The balance between (n-6) and (n-3) fatty acid availability and pathway competition between the (n-6) and (n-3) fatty acids is likely crucial to the cellular expression of a normal phenotype.

The overall objective was to investigate whether the F508 mutation in the CFTR reduces incorporation of the EFA 18:2(n-6) into phospholipids of CF cells. Results indicate that a defective CFTR reduced incorporation of 18:2(n-6) into phospholipids of CF cells. This finding provides an important insight into an aspect of the CF defect and its relationship to the EFA profile characteristic of membranes from CF cell types. The results suggest that CF is not a "true" EFA deficiency reflecting EFA intake; rather, it results from unbalanced intracellular utilization of 18:2(n-6) and perhaps other EFA metabolites. If this suggestion is correct, abnormal levels of EFA metabolites may contribute to some symptoms inherent in cystic fibrosis.

	ACKNOWLEDGMENTS

 
The authors would like to extend a sincere thanks to R. A. Frizzel for providing the transfected CF cells.

	FOOTNOTES

 
¹ Supported by the Canadian Cystic Fibrosis Foundation and the Natural Sciences and Engineering Research Council of Canada.

³ Abbreviations used: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; EFA, essential fatty acids; FAME, fatty acid methyl esters; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution; PANC-1, pancreatic adenocarcinoma cell line; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine.

Manuscript received April 6, 2000. Initial review completed July 13, 2000. Revision accepted August 10, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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