(Received for publication, January 15, 1996)
From the
The human intestinal fatty acid binding protein (IFABP) binds
long-chain fatty acids in vitro, but its intracellular
function has remained speculative. A polymorphism in the gene that
encodes IFABP results in an alanine (Ala) to threonine
(Thr
) substitution at codon 54 that alters the in
vitro binding affinity of the protein for long-chain fatty acids.
To identify potential functional variablity between Ala
and Thr
IFABP, we established permanently
transfected Caco-2 cell lines that express either Ala
or
Thr
IFABP. We found that Caco-2 cells expressing
Thr
IFABP transport long-chain fatty acids and secrete
triglycerides to a greater degree than Caco-2 cells expressing
Ala
IFABP. These results provide the first demonstration
that IFABP participates in the intracellular transport of long-chain
fatty acids. In addition, the observed increase in transport of fatty
acids across cells expressing Thr
IFABP suggests a
plausible physiologic mechanism for our prior observation that Pima
Indians with a Thr
IFABP genotype have increased
post-absorptive lipid oxidation rates and are more insulin-resistant
than Pimas with a Ala
IFABP genotype.
Long-chain free fatty acids, a major hydrolysis product of
dietary triglycerides, are absorbed from the lumen into polarized
enterocytes that line the small intestine. Following apical absorption
into the enterocytes, free fatty acids are reincorporated into
triglycerides, which are secreted basolaterally as chylomicrons. The
intestinal fatty acid binding protein (IFABP) ()is a small
(15 kDa), highly abundant protein expressed solely in enterocytes of
the proximal small intestine(1) . IFABP has been shown to bind
both saturated and unsaturated long-chain fatty acids in vitro(2, 3) . Several functions for IFABP have been
proposed, which have included the facilitation of cellular uptake
and/or transport of long-chain fatty acids within
enterocytes(1) .
We have recently reported a polymorphism in
the second exon of the FABP2 gene, which encodes human
IFABP(4) . This A G single base polymorphism results in
an alanine (Ala
) to threonine (Thr
)
substitution at amino acid 54 (codon 55). (
)Our genetic
studies with Pima Indians, a population with a high prevalence of
obesity, insulin resistance, and non-insulin-dependent diabetes
mellitus, have shown that the Thr
-encoding IFABP genotype
(frequency = 0.29) is associated with increased fasting lipid
oxidation rates and insulin resistance(4) . We further reported
that recombinant Thr
protein has a 2-fold higher affinity
for long-chain fatty acids in vitro as compared to recombinant
Ala
protein(4) .
The crystal structure of rat IFABP, which has high sequence homology to the human IFABP, has been determined(5) . The major conformational adjustment between the structure of IFABP alone and the structure of IFABP bound to fatty acid occurs at a tight turn containing residues 54 and 55. These residues shift in position when long-chain fatty acids are bound to the protein. Therefore, even a subtle change in the amino acid sequence of this turn could affect the structural properties of IFABP in such a way as to alter its ligand affinity. Since the alanine to threonine substitution at residue 54 is part of this critical turn, it is not surprising that the two forms of this protein have different affinities for long-chain fatty acids. It is also possible that this substitution could affect the kinetics of fatty acid acquisition/release to cytoplasm, transport of fatty acids across the cell, or trafficking of fatty acids to metabolic pathways within the cell.
To determine whether the
Ala
Thr substitution in IFABP alters the rate of
intracellular fatty acid transport, we analyzed fatty acid transport
across intestine-like cultured cells expressing either Ala
or Thr
IFABP. Caco-2 cells provided a model system
for analysis of lipid trafficking since these cells mimic the small
intestinal epithelium, are capable of absorbing long-chain fatty acids
and secreting chylomicrons, and do not endogenously express
IFABP(6, 7, 8, 9, 10, 11, 12) .
Introduction of Ala
and Thr
IFABP into Caco-2
cells allowed direct assessment of the effect of this single amino acid
substitution on the rate of intracellular fatty acid transport and
triglyceride release, which could potentially contribute to the
development of insulin resistance.
Apical and basolateral medium
were replaced with serum-free medium, and cells were serum-starved for
15 h. [H]Oleic acid-sodium taurocholate micelles
were prepared by mixing 100 µM sodium salt of oleic acid
(Nu-Chek Prep, Elysian, MN) and 10 µM [
H]oleic acid (DuPont NEN) with serum-free
medium containing 8 mM sodium taurocholate and incubating the
solution at 37 °C for 30 min. [
H]Palmitic
acid-sodium taurocholate micelles were prepared by the same method.
Cells were incubated with long-chain fatty acids by replacing the
apical medium with 2 ml of medium containing the fatty
acid-taurocholate micelles. The basolateral medium was replaced with
serum-free medium containing only 8 mM sodium taurocholate.
Cells were incubated with the fatty acids at 37 °C in an atmosphere
of 95% air, 5% CO
from 5 min to 48 h. At the end of the
incubation, apical and basolateral media were removed. Apical and
basolateral compartments were washed with 2 ml of phosphate-buffered
saline, and the wash was combined with the media. Scintillation fluid
was added and the radioactivity in the basolateral media was determined
by scintillation counting. Following each incubation, the monolayers
were assayed for total protein to control for potential overgrowth or
``piling-up'' of cells. All experiments were performed with
at least three filters.
Transfection of Caco-2 cells resulted in five permanently
transfected cell lines (two independent cell lines expressing
Ala IFABP and three independent cell lines expressing
Thr
IFABP), which continually exhibited identical
proliferation rates and saturation densities when maintained in media
containing 500 µg/ml G418. For lipid transport studies, these cells
were grown on filter supports until 14 days post-confluence to best
mimic mature enterocytes(8) . Tight monolayers of cells
(transepithelial resistances between 250 and 320 ohms/cm
)
consistently contained equivalent amounts of total protein (<10%
variability). Mature, tight monolayers of cells were incubated for 1.5
h in media containing 100 µM [
H]oleic acid or
[
H]palmitic acid in taurocholate
micelles(11) . Following each incubation,
H-lipid
that had been transported across the transfected cell monolayer and
secreted into the basolateral media was measured. For both oleic acid
and palmitic acid, all three cell lines expressing Thr
IFABP transported more long-chain fatty acids than either of the
cell lines expressing Ala
IFABP (Table 1). Moreover,
in all of the transfected cell lines, incubation with palmitic acid
resulted in greater lipid transport than incubation with oleic acid.
Western blot analysis revealed variable expression of IFABP among
these transfected clones (Table 1). However, cells from clones
Ala B and Thr
A contained nearly identical
levels of IFABP mRNA, as determined by reverse transcription/PCR
amplification of IFABP cDNA, and consistently produced equivalent
amounts of Ala
or Thr
IFABP, as determined by
quantification of the 15-kDa IFABP immunoreactive band on three
occasions over the period of several months (Fig. 1). Therefore,
these two clones were compared in all further studies.
Figure 1:
Detection of IFABP mRNA and
protein in transfected Caco-2 cells from clones Ala B and
Thr
A. A, poly(A) RNA from transfected and
non-transfected Caco-2 cells was reverse-transcribed. The resulting
cDNA was used as a template for 25 cycles of PCR using primers that
span 300 base pairs of IFABP cDNA. B, Western blot analysis of
protein lysates from these clones using antiserum raised against rat
IFABP. Human IFABP migrates at 15 kDa.
Transfected
cells were incubated for 3.5, 24, or 48 h in media containing
[H]oleic acid or
[
H]palmitic acid in taurocholate micelles. At all
time points, and with both the saturated (palmitic acid) and
unsaturated (oleic acid) fatty acids, approximately twice as much
H-lipid was apically to basolaterally transported across
the Thr
cells as compared to the Ala
cells (Fig. 2, A and B). The difference in rate of
apical to basolateral transport of long-chain fatty acid across the
Ala
and Thr
cells appears to be specific for
molecules that interact with IFABP, since no difference in
[
C]glucose transport was observed in these cells (Fig. 2C). This increase in lipid transport was not due
to an increased level of endogenously expressed LFABP in the
Thr
-expressing Caco-2 cells, as LFABP may also contribute
to the total transport of long-chain fatty acids. However, Caco-2 cells
expressing either Ala
or Thr
IFABP were found
to produce lower levels of LFABP than non-transfected Caco-2 cells.
Lower levels of LFABP, a maturation-dependent protein, were observed in
transfected cells at both 4 days and 14 days post-confluence, when
compared to non-transfected cells ( Fig. 3and Table 2),
which suggests a co-regulation between the intestinal and liver forms
of this protein. Since LFABP was more abundant in the non-transfected
cells than comparably mature IFABP transfected cells, non-transfected
cells did not provide a ``base-line'' control for lipid
transport by IFABP in these experiments (Fig. 4). The
contribution of LFABP to total transport in the IFABP transfected cells
remains unclear. At 4 days post-confluence, when LFABP is barely
detectable in the transfected cells, a 2-fold difference in transport
across Ala
and Thr
cells is observed. At 14
days post-confluence, a similar 2-fold difference in transport is
observed, despite the greater abundance of LFABP in these mature cells.
These results support a mechanism whereby LFABP and IFABP do not
transport in a simple additive manner, since the total amount of FABP (i.e. liver and intestinal) is not proportional to the amount
of transport.
Figure 2:
Transport of H-lipid and
[
C]glucose across transfected Caco-2 cells from
clones Ala
B and Thr
A. Panel A,
apical exposure of the cells to 100 µM of
[
H]oleic acid (specific activity 1 nmol/83,000
dpm) in sodium taurocholate micelles. Cells were incubated for 3.5 and
24 h, and the basolateral medium was analyzed for
H. At
each time point, three or four replicate filters were analyzed for each
cell type. The mean ± S.D. is given for three separate
experiments. Panel B, apical exposure of the cells to 100
µM [
H]palmitic acid (specific
activity 1 nmol/83,000 dpm) in sodium taurocholate micelles. Cells were
incubated for 24 and 48 h, and the basolateral medium was analyzed for
H. At each time point, three replicate filters were
analyzed for each cell type. The mean ± S.D. is given for three
separate experiments. Panel C, apical exposure of the cells to
5.68 mM [
C]glucose (specific activity 1
nmol/200,000 dpm). Cells were incubated for 3.5 and 24 h, and the
basolateral medium was analyzed for
C. The mean ±
S.D. is given for four replicate filters done in one
experiment.
Figure 3:
Differential expression of liver FABP in
transfected and non-transfected Caco-2 cells. Western blot analysis,
using an antibody immunoreactive with human liver FABP, detected
greater amounts of liver FABP in non-transfected Caco-2 cells than in
transfected Caco-2 cells. This was observed in both immature Caco-2
cells (4 days post-confluent) as well as mature Caco-2 cells (14 days
post-confluent). In addition, mature cells expressing Thr IFABP contain slightly less liver FABP than cells expressing
Ala
IFABP.
Figure 4:
Rates of basolateral lipid secretion
across Ala and Thr
transfected cells, and
non-transfected Caco-2 cells. Cells were apically exposed to a mixture
of 50 µM [
H]oleic acid and 50
µM [
H]palmitic acid in sodium
taurocholate micelles. At each time point, the basolateral medium was
analyzed for
H.
The nature of the basolaterally secreted H-lipids components was analyzed by thin-layer
chromatography. Following 24 h of apical incubation with
H-oleate, the predominate lipids isolated from the
basolateral media were triglycerides and free fatty acids. Similar
levels of free fatty acids were identified in the basolateral media
from Ala
and Thr
cells. In contrast,
triglyceride secretion differed, where Thr
cells secreted
a 5-6-fold greater amount of triglyceride compared to Ala
cells (Fig. 5). Secretion of cholesterol esters, although
representing a small percent of total lipid, also consistently differed
between the Ala
and Thr
cells (1.4% and 6% of
the total secreted lipids for Ala
and Thr
,
respectively).
Figure 5:
Secretion of H-triglycerides
from Caco-2 cells expressing Ala
or Thr
IFABP. Cells were apically incubated with
[
H]oleate for 24 h. Lipids were extracted from
the basolateral media and separated by thin-layer chromatography. The
mean ± S.D. for the amount of
H-lipid that migrated
as triglyceride is given for three experiments, where each experiment
contained four replicate filters.
The differences in fatty acid transport across
Ala and Thr
transfected Caco-2 cells provides
the first direct evidence that IFABP participates in the intracellular
transport of dietary long-chain fatty acids in vivo. The
mechanism of movement of fatty acids into enterocytes remains
speculative. Fatty acids may require a membrane transport protein or
alternatively, may enter a cell by passive diffusion. Putative fatty
acid transport proteins have been identified in liver, heart, skeletal
muscle, and
adipocytes(14, 15, 16, 17) .
Hamilton et al.(18) have demonstrated that fatty acid
movement into clonal pancreatic B-cells, as well as phospholipid
bilayer vesicles, occurs predominantly via passive diffusion of the
non-ionized form across the plasma membrane rather than via a membrane
protein carrier. If long-chain fatty acids enter enterocytes via
diffusion, then any mechanism that increases the shuttling of fatty
acids away from the inner membrane surface would drive the diffusion
gradient toward greater uptake. We did not observe a significant
difference in the cell-associated fatty acids in Ala
and
Thr
Caco-2 cells at 3.5 h (57-60% and 54-58%
of total radiolabel added apically) or at 24 h (77-85% and
79-83% of total radiolabel added apically), but it remains
undetermined whether these fatty acids have been intracellularly
absorbed (uptake) or whether a large portion remain membrane-bound. Our
data confirmed that Caco-2 cells retain approximately 90% of their
lipids intracellularly at 24 h post-incubation(12) , yet we
still observed that increasing the concentration of fatty acid
presented apically to the transfected Caco-2 cells resulted in a net
increase in lipid secretion from Thr
compared to
Ala
cells at 24 h (Table 3). Since dietary fat is
usually ingested as a bolus, and intestinal absorption rates are very
high (approximately 96%) and remarkably constant among
individuals(19) , a high concentration of long-chain fatty
acids within an enterocyte would be predicted following a high fat
meal. Therefore, the effect of the Ala
Thr
substitution on lipid transport and secretion may be greatest
immediately following a high fat meal.
Previous studies have
indicated a direct relationship between levels of insulin resistance
and increased concentrations of circulating free fatty acids and
triglycerides(20, 21, 22, 23, 24, 25, 26, 27) .
Free fatty acids normally provide an alternative fuel source to glucose
for energy during periods of fasting. Increased concentrations of
plasma free fatty acids inhibit glucose uptake in a dose-dependent
fashion (25) and cause insulin resistance in target tissues
such as muscle, and possibly stimulate increased insulin release from
pancreatic -cells(26) . Alternatively, increased
concentrations of triglycerides, independent of circulating free fatty
acids, can also affect glucose metabolism. Triglycerides alone account
for at least 25% of the total decrease of forearm glucose uptake and
glucose oxidation following Intralipid infusion(27) . The
effect of triglycerides on carbohydrate metabolism seems to follow the
same intracellular pathway as free fatty acids, since triglyceride
hydrolysis increases the intracellular pool of free fatty acids,
thereby enhancing
-oxidation and decreasing glucose
utilization(27) .
If Thr IFABP also increases
fatty acid transport and triglyceride release in intact jejunum, then
individuals who express the Thr
IFABP genotype would be
predicted to: 1) release intestinally absorbed dietary fatty acids to
the lymph at a faster rate than individuals with the Ala
genotype and 2) process more dietary long-chain fatty acids into
chylomicron triglycerides than individuals who express the Ala
IFABP genotype. Either or both of these processes would result in
decreased rates of insulin-mediated glucose uptake and increased rates
of insulin release from pancreatic
-cells, consistent with the
observed insulin resistance and hyperinsulinemia found in subjects with
the Thr
IFABP genotype(4) . Of particular
relevance is a comparison of the insulin responses to oral glucose (75
g) versus a mixed meal (20% protein, 40% carbohydrate, 40%
fat) in subjects with the Ala
and Thr
IFABP
genotypes. Although insulin responses for both of these studies were
significantly greater in subjects with the Thr
genotype, a
much greater difference between the two IFABP genotypes was observed
following the mixed meal, which contained dietary fats. This is
consistent with the results presented in the Caco-2 model system.
However, further studies are needed to determine whether a more rapid
rate of release of dietary lipid to circulation, or differential
esterification of absorbed fatty acids, or both mechanisms, are
actually contributing factors to the insulin resistance and
hyperinsulinemia observed in individuals with the Thr
IFABP genotype. Direct measurements of absorption levels and
oxidation rates of ingested long-chain fatty acids are currently being
analyzed in Pimas homozygous for the Ala
- or the
Thr
-encoding IFABP alleles.