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F508 Mutation in the Cystic Fibrosis Transmembrane Conductance Regulator Alters Control of Essential Fatty Acid Utilization in Epithelial Cells1
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 |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
|
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.
|
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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
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), implying that the
effect of the CFTR on 18:2(n-6) incorporation into phospholipids
observed in the present study may be related to chloride conductance.
The lack of difference in the chloride conductance in control vs. CF
cell types in this study suggests that the incorporation of 18:2(n-6)
is independent of the role of the CFTR as a chloride channel. Chloride
channel blockers function differently than the CFTR. Thus, it is also
possible that the effect of the blocker on fatty acid incorporation was
related to factors such as chloride concentration rather than chloride
conductance (Kang et al. 1992
). The possibility also
exists that the CFTR regulates other unidentified cellular functions,
thereby exerting effects on 18:2(n-6) incorporation into complex
lipids. The importance of a functional CFTR for the normal
intracellular utilization of 18:2(n-6) is evident (Fig. 3)
. Whether
this role is related to the chloride channel function or due to
alterations in other functions of the CFTR or in undefined functions
mediated by the CFTR is not known.
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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