 |
INTRODUCTION |
Under normal conditions 60-70% of the oxidative metabolism of
the heart comes from the
-oxidation of fatty acids (1). Extraction
of fatty acids complexed to albumin by the heart is very efficient,
with as much as 70% of the
LCFAs1 from the blood
entering the myocytes during a single transition through the cardiac
capillary system (2). Albumin-bound fatty acids are generated in the
heart from triglycerides contained in lipoprotein particles by the
action of lipases. Lipoprotein lipase is bound to the luminal face of
endothelial cells after secretion from the parenchymal cells of the
heart (3). Unlike hepatocytes, which have direct contact with the
blood, fatty acids in the heart must first traverse the endothelial
cell barrier. It is unclear how the LCFAs in the blood are transported
across the endothelial cells, but it is unlikely that the entire
LCFA-albumin complex leaves the circulation, because albumin does not
diffuse through the clefts between cardiac vascular endothelial cells in significant amounts (4). Once in the interstitial fluid between
endothelial cells and myocytes, fatty acids are again bound to albumin
and rapidly taken up by the muscle cells. Although it was originally
believed that uptake of LCFAs into cardiac myocytes is purely mediated
by diffusion across the sarcolemma (4, 5), later work has shown that
transport is predominantly protein-mediated (6-9). A fatty acid
transport protein (FATP) was identified by expression cloning from a
murine adipocyte cDNA library as a protein that facilitates the
uptake of LCFAs when overexpressed in adipocytes (9). This protein,
later renamed FATP1 (10), is induced during adipocyte differentiation
in vitro and is expressed in fat tissue and in the brain,
heart, kidneys, and skeletal muscle but not the liver. Subsequently, we
reported the discovery of a large family of FATPs characterized by the
presence of an FATP signature sequence of 311 amino acids that is
highly conserved among FATP family members (10, 11). FATP expression
patterns include proteins exclusively or predominantly expressed in the
liver, kidneys, small intestine, or in white adipose tissue (WAT). All FATPs are integral membrane proteins (12, 13), and detailed subcellular
localization studies have been reported for FATP1 and FATP4. The
intestinal FATP, FATP4, is localized to the apical side of enterocytes
and mediates the uptake of dietary fatty acids (14). FATP1 is present
in the heart but is also strongly expressed in adipose tissue where its
subcellular localization can change on insulin stimulation from an
intracellular perinuclear compartment to the plasma membrane (15). The
mechanisms and requirements for LCFA uptake through FATPs are poorly
understood. Acyl-CoA synthetase activity has been demonstrated for
several FATPs (16, 17), and it has been suggested that this activity is
required for transport. This notion has been challenged, however, by
the recent observation that catalytic and transport activities of the
yeast FATP homologue, FAT1, can occur independently (18).
In addition to FATPs, two other proteins have been implicated in
myocardial LCFA uptake: the cytoplasmic heart-specific fatty acid-binding protein and CD36. The strongest support for the
role of these proteins comes from data demonstrating that in both
heart-specific fatty acid-binding protein (6) and CD36 (19) null mice
cardiac fatty acid uptake and use is substantially reduced.
Here we show that a novel member of the FATP family, FATP6, is a
functional fatty acid transporter with a heart-specific expression pattern. Further, FATP6 localizes to the sarcolemma of cardiac myocytes
juxtaposed with blood vessels where it colocalizes with CD36. This fact
suggests that a novel mechanism may exist for the uptake of LCFAs into
cardiac myocytes using FATP6, thus potentially implicating the molecule
in a broad range of physiological and pathological cardiac functions.
 |
EXPERIMENTAL PROCEDURES |
Fatty Acid Uptake Assays--
Clones encoding human FATP6 were
identified by a search of public data bases for sequences similar to
murine FATP1-5 coding regions using the BLASTX algorithm (20). A DNA
fragment containing the entire hsFATP6 coding sequence was inserted
into the mammalian expression vectors pMet7 or pIRES-neo
(Clontech) for transient and stable transfections,
respectively. Transient transfection assays were performed as described
previously (10). For stable transfections, cells that had taken up the
DNA were selected with 1 mg/ml G418 (Invitrogen). BODIPY-fatty
acid uptake assays using a fluorescence-activated cell sorter
and 14C-labeled fatty acid uptake assays were performed as
described previously (10, 14).
In Situ Hybridization--
Human heart samples were obtained
from the University of Pittsburgh Medical Center. In situ
hybridizations were performed essentially as described previously (14).
Briefly, paraformaldehyde-fixed dehydrated sections were hybridized
with 35S-radiolabeled (5 × 107 cpm/ml)
cRNA probes generated from the open reading frame of FATP6. After
hybridization, slides were washed and dehydrated. To detect the
localization of mRNA transcripts, the slides were dipped in Kodak
NBT-2 photoemulsion and exposed for 7 days at 4 °C followed by
development with Kodak Dektol developer. Slides were counterstained
with hematoxylin and eosin and photographed. Controls for the in
situ hybridization experiments included the use of a sense probe,
which showed no signal above background in all cases.
Northern Blotting--
Human mRNA blots were obtained from
Clontech. Blots were probed with
32P-labeled DNA probes generated by PCR from the
5'-untranslated region of FATP6 using the Rapid-Hyb buffer (Amersham
Biosciences).
Immunofluorescence Microscopy--
Unfixed rhesus monkey or
mouse heart was washed with Hanks' buffered salt solution containing 1 mM EDTA, infused with 2.3 M sucrose solution,
and embedded in O.C.T. 4583 compound. The material was cut into
thick sections (15-40 µm). The sections were washed in
phosphate-buffered saline containing 1% bovine serum albumin, 10%
fetal calf serum, and 1% normal donkey serum to block nonspecific
binding. Primary and secondary antibodies were diluted in blocking
solution and incubated for 1 h. The sections were mounted in 90%
glycerol/phosphate-buffered saline containing 1 mg/ml
paraphenylinediamine and examined with a Zeiss LSM10 confocal system.
Antibodies--
Anti-FATP6 serum was raised by immunization of
rabbits with the last 90 C-terminal amino acids fused to GST. Serum was
affinity-purified over protein A columns and found to have minimal
cross-reactivity against other FATP family members. Monoclonal
anti-caveolin 3 antibodies and anti-CD31 were purchased from BD
Biosciences. The anti-CD36 monoclonal antibody was a generous gift from
Dr. Maria Febbraio.
 |
RESULTS |
Cloning and Chromosomal Localization of FATP6--
We initially
reported the 3' sequence of a sixth human FATP gene using BLAST
screens of FATP1 against the NCBI public data bases (10). Using the
known 3' FATP6 sequence, we screened public data bases of human
expressed sequence tags and identified a full-length cDNA for
FATP6. The cDNA clone was confirmed by sequencing and was found
later to be identical to a cDNA sequence deposited in GenBankTM as VLCS-H1 (AF064254). The FATP6 cDNA
encodes for a 619-amino acid protein with a predicted molecular weight
of 70.1 kDa. Alignments of the full-length FATP6 protein with the
full-length sequences of all other known human FATPs were done using
the ClustalW algorithm. Fig.
1A shows that FATP6 is a
member of the FATP family and is most closely related to FATP2 (51.1%
identity). FATP6 is part of a larger group including FATP5 and FATP3
that is clearly distinct from the FATP1 and FATP4 genes (Fig. 1). Using
radiation hybrid mapping, we localized hsFATP6 to human chromosome 5q23
(D5S1896). hsFATP6 sequence and localization was also
confirmed by the human genome project, which showed that the coding
region of the gene is distributed over 10 exons spanning 67 kb on
chromosome 5q. Using alignments of hsFATP6 against the translated mouse
genome, we were able to identify a murine homologue (mmFATP6). The
mmFATP6 protein is 78% identical to hsFATP6 and is also encoded in
10 exons spanning 55.6 kb on mouse chromosome 18.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 1.
FATP6 is a heart-specific FATP. A,
alignment of human FATPs. Full-length protein
sequences of all six human FATPs were aligned using the Clustal
algorithm in the DNA Star MegAlign program. The calculated percent
identity is shown. Full-length FATP sequences were independently
aligned using ClustalX to generate a phylogenetic tree, which was
plotted using TreeViewPPC. The bar indicates the number of
substitutions per residue with 0.1 corresponding to a distance of 10 substitutions/100 residues. B, FATP6 expression
pattern. Northern blot analysis of human FATP6 expression
was performed using human tissue poly(A) mRNA blots
(Clontech). The probe was generated from the
5'-untranslated region of hsFATP6. C, tissue
Western blot. Equal amounts of mouse organ lysates (10 µg/lane) were separated by electrophoresis on 8-16% gradient gels,
blotted, and probed with an FATP6-specific antiserum. The apparent
molecular mass of the FATP6 signal is 70 kDa.
|
|
FATP6 Expression Is Heart-specific--
An FATP6-specific probe
from the 5'-untranslated region of the gene was used to detect its
expression pattern using Northern blot analysis of human tissue
mRNAs. FATP6 was strongly expressed in heart but was absent from
the lung, spleen, brain, rectum, colon, liver, muscle, stomach, ileum,
jejunum, and pancreas (Fig. 1B). Moderate levels of FATP6
(approximately 20-40 times lower than in the heart by densitometric
quantification of Northern blot data) were found in the placenta (Fig.
1B), testis, and adrenal glands (data not shown). FATP6
mRNA was present at very low levels in kidney, bladder, and uterus
(Fig. 1B). A tissue Western blot of 12 murine organs
revealed that mmFATP6 is predominantly expressed in the heart in this
species (Fig. 1C). Weak mmFATP6 expression was observed in
the testis (Fig. 1C).
FATP6 Is Expressed by Cardiac Myocytes--
To identify the cell
type that expresses FATP6, in situ hybridizations were
performed with human heart sections. A sense probe showed only a
very low background signal (Fig.
2A), whereas the corresponding
FATP6-specific antisense probe showed a strong signal with silver grain
accumulations over cells identified by morphology as cardiac myocytes
(Fig. 2B).

View larger version (165K):
[in this window]
[in a new window]
|
Fig. 2.
FATP6 is localized in cardiac myocytes
by in situ hybridization. Dark field (left
panels) and phase contrast (right panels) of in
situ hybridization of human heart sections with either a sense
control (A) or with an FATP6-specific antisense probe
(B and C) is shown. Bar represents 200 µM.
|
|
Localization of the FATP6 Protein to Cardiac Plasma Membrane
Specializations--
To further study the subcellular localization of
FATP6, we raised an antiserum against the C-terminal portion of FATP6.
Sections of rhesus monkey hearts were co-incubated with anti-FATP6
serum and anti-caveolin 3 antibodies (Fig.
3). Caveolin 3 is highly expressed by
cardiac myocytes and was used as a plasma membrane marker (21).
Confocal microscopy showed FATP6 in virtually all cardiac myocytes
(Fig. 3, middle panels). Staining was FATP6-specific, because preincubation of the FATP6 and caveolin 3 antisera with the
FATP6 fusion protein antigen abolished FATP6 but not caveolin 3 staining (Fig. 3A). At higher magnifications (Fig. 3,
C and D), FATP6 protein was found almost
exclusively on the sarcolemma. Unlike the caveolin 3 staining, however,
FATP6 was not evenly distributed along the membrane but was
concentrated in shorter segments. Myocytes derive most of their LCFA
from the interstitial space adjacent to the microcapillaries. To test
whether FATP6 was concentrated on sarcolemma sections in the direct
vicinity of blood vessels, we costained heart sections with FATP6 and a marker for endothelial cells, CD31 (Fig.
4, left panels). FATP6 expression was clearly observed in the areas directly adjacent to the
microvasculature, whereas expression was absent in the areas of larger
blood vessels (Fig. 4, A and B). This expression pattern was apparent at higher magnifications as shown in Fig. 4C, where a small branch of a blood vessel is surrounded by
FATP6 expression on the myocytes above and below the blood vessel. This expression pattern is consistent with a role of FATP6 in the uptake of
LCFAs from the interstitial space into cardiac myocytes.

View larger version (108K):
[in this window]
[in a new window]
|
Fig. 3.
FATP6 is localized on the sarcolemma.
Fresh-frozen rhesus monkey heart sections were stained with either
anti-caveolin 3 antibodies (left panels) or FATP6- specific
anti-serum (middle panels), and an overlay projection is
shown in the right panels. A, caveolin 3 and
FATP6 antisera were preincubated with FATP6-GST fusion proteins. The
bar represents 20 µM. B and
C, sections were stained with caveolin 3 and FATP6 antisera.
The bar represents 20 µM. D,
sections were stained with caveolin 3 and FATP6 antibodies.
Bar represents 5 µM.
|
|

View larger version (112K):
[in this window]
[in a new window]
|
Fig. 4.
FATP6 is localized adjacent to blood
vessels. Fresh-frozen rhesus monkey heart sections were either
stained with anti-CD31 antibodies (left panels) or an
FATP6-specific antiserum (middle panels), and an overlay
projection is shown in the right panels. The bar
represents 20 µM in A and B and 5 µM in C.
|
|
FATP6 Mediates the Uptake of Long Chain Fatty Acids--
To
confirm that FATP6, like the other FATP family members, facilitates the
uptake of long chain fatty acids, we transiently transfected COS
cells with mammalian expression vectors containing either FATP1, FATP4,
or FATP6 into 293 cells (Fig.
5A). Transfected cells were
identified by CD2 cotransfection, and uptake of BODIPY-labeled LCFA was
determined as described previously (10, 14). Stable cell lines
overexpressing FATP6 were generated by the transfection and selection
of 293 cells. Initially, approximately 30 independent FATP6 clones were
isolated. However, the cell lines with the highest uptake rates lost
the ability to take up fatty acids after a few generations possibly
because of fatty acid toxicity. Uptake of 14C-labeled
oleate by one of the stable FATP6 cell lines with intermediate uptake
rates is shown in Fig. 5B along with uptake by cells stably expressing FATP1 or the expression construct alone. Fig. 5,
A and B, demonstrates that overexpression of
FATP6 can indeed increase uptake of LCFAs. We further tested the
substrate preference of hsFATP6 in comparison with hsFATP4 and
hsFATP1 using stably transfected cell lines (Fig.
5C). Interestingly, FATP6 and FATP1 had a higher preference
for palmitate and linoleate compared with FATP4, the major intestinal
LCFA transporter (Fig. 5C). As expected, none of the FATPs
tested increased the uptake of fatty acids with chain length shorter
than C10 (Fig. 5C).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
FATP6 transports long chain fatty acids.
A, 293 cells were cotransfected with mammalian expression
vectors pCDNA-CD2 either alone (control) or in combination with one
of the FATP-containing expression vectors (pMET7-hsFATP1,
pMET7-hsFATP4, or pMET7-hsFATP6). BODIPY-fatty acid uptake assays were
performed as described under "Experimental Procedures."
Bars represent the mean BODIPY-fatty acid fluorescence of
CD2-positive cells. B, the rate of 14C-oleate
uptake by 293 cells, stably expressing either hsFATP6
(diamonds) or hsFATP1 (circles), was compared
with that of cells containing the vector with no insert
(squares). C, specific uptake of different fatty
acid substrates by 293 cell lines stably expressing human FATP6, FATP4,
or FATP1. The uptake rates in pmol/min/million cells after subtraction
of background are shown. Background uptake was measured using a 293 cell line that stably expressed pcDNA 3.1 without inserted
cDNA. Uptake of palmitate, oleate, linoleate, and octanoate by the
control cell line was 564, 662, 640, and 0 pmol/min/million cells,
respectively. All fatty acids were at a 50 µM final
concentration.
|
|
FATP6 Is the Predominant FATP in the Heart--
Because both FATP1
and FATP6 are expressed in the heart, we wanted to compare their
overall abundance. To this end, we performed Western blots with equal
amounts of WAT and heart lysates from mice next to titrations of known
amounts of FATP1 or FATP6 antigen. The blots were probed with FATP1 and
FATP6 sera, respectively, and signals were quantitated by densitometry.
FATP6 was absent from WAT but was robustly expressed in the heart.
FATP1 was more than 10 times more abundant in WAT than in the heart
(Fig. 6). After calibrating the Western
blot signals to the antigen titrations, we were able to directly
compare the FATP1 and FATP6 signals in the heart (Fig. 6B).
FATP1 was expressed at ~100 pg/ml heart lysate (1 mg/ml total
protein), whereas FATP6 was more than 20 times (2139 pg/mg lysate) more
abundant (Fig. 6B), which indicates that FATP6 is the
predominant FATP in the heart.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 6.
FATP6 is the predominant FATP in the heart.
A, the indicated amounts of FATP1-GST fusion protein
(upper row), FATP6-GST fusion protein (lower
row), mouse WAT, or mouse heart were separated by PAGE and blotted
with FATP6 or FATP1 antibodies. All blots were exposed on the same
film. B, ECL signal strength of the GST fusion titrations
for FATP1 and FATP6 were determined by densitometry and plotted as a
function of protein amount (not shown). Linear regressions from these
plots were used to calculate protein amounts for FATP1 and FATP6 in the
adipose tissue and heart lysates expressed as picogram of antigen per
milligram of lysate.
|
|
FATP6 Colocalizes with CD36 on the Myocyte Membrane--
Because
both FATP6 and CD36 are plasma membrane proteins that have been
implicated in the uptake of LCFAs into cardiomyocytes, we wanted to
test a possible interaction between these proteins. To this end, we
stained sections of mouse hearts with antibodies against FATP6
(red channel), CD36 (green channel), caveolin 3 (blue channel) and a DNA-specific dye
(4',6-diamidino-2-phenylindole, pink channel). As in
sections of primate hearts, murine FATP6 was present in subregions of
the plasma membrane (Fig. 7,
A, panel I and B, panel I).
Staining of the same sections with an anti-CD36-specific monoclonal
serum showed a similar staining pattern (Fig. 7, A, panel II, and B, panel II), with
streaks on the sarcolemma and pronounced globular aggregates (Fig. 7,
white arrows). In contrast, caveolin 3 was evenly
distributed along the plasma membranes of all myocytes (Fig. 7,
A, panel III, and B, panel
III). The overlay of FATP6 and CD36 signals (Fig. 7, A,
panel IV, and B, panel IV) showed
that FATP6 was distributed somewhat more broadly along the sarcolemma.
Significant colocalization of FATP6 with CD36 was most apparent in the
globular aggregates (Fig. 7, white arrows). The overlay of
FATP6, CD36, and caveolin staining (Fig. 7, A, panel
V, and B, panel V) showed further that
all three molecules are present in these membrane specializations (Fig.
7, white arrows) as indicated by the appearance of the white
areas. These data provide evidence suggesting that members of the FATP
family may interact with CD36.

View larger version (111K):
[in this window]
[in a new window]
|
Fig. 7.
FATP6 partially colocalizes with CD36.
Thin sections of mouse hearts were stained for FATP6 (I),
CD36 (II), and caveolin 3 (III). Two different
magnifications (A and B) are shown
(bars represent 10 µM). Colocalization between
FATP6 and CD36 shows as yellow in the superimposition of
sections I and II. Superimposition of
all three channels without (V) or with a bright field image
(VI) shows colocalization of FATP6, CD36, and caveolin 3 (arrows) in the white areas.
|
|
 |
DISCUSSION |
Fatty acids are a major source of energy for cardiac myocytes, and
changes in fatty acid metabolism have been implicated in cardiac
disease. The mechanism by which fatty acids from the interstitial space
enter myocytes is not well understood, but it has been proposed that
the bulk of this uptake occurs via protein-mediated transport (6-9).
Here we report the cloning and characterization of a novel member of
the fatty acid transport protein family, termed FATP6, in humans and
mice. Heart muscle expresses other FATP family members, most notably
FATP1 as well as FATP6. FATP6 is heart-specific, however, whereas FATP1
is expressed in a variety of other organs including adipocytes,
skeletal muscle, and the brain (9, 10, 22). Further supporting the
notion that FATP6 is the predominant cardiac FATP is our finding that
the protein is more than 20 times more abundant than FATP1 in mouse
heart lysates. The heart also has a very distinct uptake pattern of
fatty acids and shows, in contrast with WAT, a preference for palmitate
compared with oleate (23). Interestingly, this trend is reflected by
the LCFA uptake pattern of the FATP6-stable cell line, which is
consistent with the idea that a significant part of the cardiac LCFA
uptake is mediated by FATP6.
Amino acid sequence comparisons of the FATP family show that human
FATP6 is most closely related to hsFATP2, a fatty acid transporter
predominantly expressed in the liver and kidneys. FATP2 is the
homologue of a rat gene previously identified by others as a very long
chain acyl-CoA synthase (24). This notion was subsequently challenged
by the finding that FATP2 can function as a fatty acid transporter
(10). It is unlikely that FATP6 is a peroxisomal very long chain
acyl-CoA synthase, because our immunofluorescence studies of FATP6
distribution in cardiac myocytes clearly demonstrated that FATP6 is
almost exclusively localized to the plasma membrane of cells. However,
the mechanism by which FATPs transport LCFAs across phospholipid
bilayers is poorly understood, and long chain as well as very long
chain acyl-CoA activities have been demonstrated for several mammalian
FATP and yeast FATP (16, 17). Therefore, the possibility that uptake is
coupled to CoA activation of LCFA, either directly by FATPs or by a
closely associated long-chain acyl-CoA synthetase, cannot be
excluded (13). In this context, it is noteworthy that recent studies of
the yeast FATP homologue FAT1 (18) have demonstrated that specific
mutations in FAT1 can distinguish the fatty acid import from the very
long chain acyl-CoA synthetase activities. Zou et al. (18)
noted that two mutations (S258A and D508A) greatly diminished long
chain and very long chain acyl-CoA synthesizing activity and that these
residues are conserved in FAT1 and FATPs 1 through 5. Alignment of
murine and human FATP6 sequences with the other family members shows
that both residues are also conserved in FATP6 (data not shown), which
suggests that these mammalian proteins could also function as LCFA
transporters in the absence of catalytic activity. Most importantly,
direct measurements of long chain (C16:0) and very long chain (C24:0)
acyl-CoA activity of hsFATP6 by Steinberg et al. (25) showed
no activity above background when the protein was overexpressed in
COS-2 cells. Combined with the data presented here, this finding
demonstrates clearly that acyl-CoA activity is not required for
FATP6-mediated LCFA uptake.
FATP6 is exclusively targeted to areas of the sarcolemma that are
directly juxtaposed to microvasculature. This characteristic of FATP6
contrasts with the insulin-sensitive glucose transporter Glut4, which
on translocation to the sarcolemma is evenly distributed on the plasma
membrane (26), and possibly reflects the faster diffusion rate of
glucose compared with albumin-bound LCFAs.
Although FATP overexpression alone leads to an increase in LCFA uptake,
it is likely that in vivo several proteins interact to
facilitate efficient uptake of fatty acids in the heart. In addition to
FATPs, several other proteins have been implicated in LCFA uptake by
cardiac myocytes, most notably CD36 and fatty acid-binding proteins,
and it has been suggested that these proteins may interact to
facilitate efficient LCFA uptake (13). Here we demonstrate for the
first time that FATPs are in close physical proximity to CD36 on the
plasma membrane. This fact supports the idea that scavenger receptors
may help to sequester LCFAs on the plasma membrane and subsequently
pass them on to FATPs for uptake.
Changes in heart fatty acid metabolism have been linked to various
cardiac disorders, especially cardiac hypertrophy (27) and ischemic
injury (28). Although normal hearts use fatty acids as their primary
energy source, hearts of patients with cardiac hypertrophy rely on
glucose metabolism accompanied by a dramatic decrease in fatty acid
oxidation (27, 29). Deletion of the genes involved in fatty acid use in
the heart has in fact been shown to cause cardiomyopathy (6, 30). Fatty
acid use can interfere with recovery from ischemic injury, and agents
that block fatty acid oxidation, such as etomoxir and dichloroacetate, improve the recovery of contractile function after ischemic injury in
rats (28). A recent study comparing FATP1 expression levels in healthy
hearts with hearts from patients with cardiomyopathy found no changes
in mRNA levels for this transporter (31). FATP6 expression was not
measured, however. It will be of great interest to determine whether
FATP6 is altered during the development of cardiac abnormalities and
whether overexpression or inhibition of the transporter can affect
cardiac disease.