Membrane Fatty Acid Transporters as Regulators of Lipid Metabolism: Implications for Metabolic Disease

TLDR: In this article, it was shown that following an acute stimulus, particularly insulin or muscle contraction, specific fatty acid transporters translocate from intracellular stores to the plasma membrane to facilitate fatty acid uptake.

Abstract: Long-chain fatty acids and lipids serve a wide variety of functions in mammalian homeostasis, particularly in the formation and dynamic properties of biological membranes and as fuels for energy production in tissues such as heart and skeletal muscle. On the other hand, long-chain fatty acid metabolites may exert toxic effects on cellular functions and cause cell injury. Therefore, fatty acid uptake into the cell and intracellular handling need to be carefully controlled. In the last few years, our knowledge of the regulation of cellular fatty acid uptake has dramatically increased. Notably, fatty acid uptake was found to occur by a mechanism that resembles that of cellular glucose uptake. Thus, following an acute stimulus, particularly insulin or muscle contraction, specific fatty acid transporters translocate from intracellular stores to the plasma membrane to facilitate fatty acid uptake, just as these same stimuli recruit glucose transporters to increase glucose uptake. This regulatory mechanism is important to clear lipids from the circulation postprandially and to rapidly facilitate substrate provision when the metabolic demands of heart and muscle are increased by contractile activity. Studies in both humans and animal models have implicated fatty acid transporters in the pathogenesis of diseases such as the progression of obesity to insulin resistance and type 2 diabetes. As a result, membrane fatty acid transporters are now being regarded as a promising therapeutic target to redirect lipid fluxes in the body in an organ-specific fashion.

Figures

FIG. 1. A: putative molecular mechanism(s) for the cellular uptake of long-chain fatty acids (FA) and of very-long-chain fatty acids (VLCFA). 1: In view of their hydrophobic nature, fatty acids could dissociate from their albumin binding sites and cross the plasma membrane by simple diffusion (referred to as uptake by passive diffusion). 2: Alternatively, membrane-associated proteins, such as the peripheral membrane protein FABPpm or the transmembrane protein CD36, could act, either alone or together, as acceptor for fatty acids to increase their concentration at the cell surface and thus enhance the number of fatty aciddiffusion events. 3: CD36 itself may also facilitate the transport of fatty acids across the phospholipid bilayer (uptake by facilitated diffusion). Once at the inner side of the cell membrane, fatty acids are bound by cytoplasmic FABP (FABPc) before entering metabolic or signaling pathways. 4: Additionally, a minority of fatty acids are thought to be transported by FATP1 and rapidly activated by plasma membrane acyl-CoA synthetase (ACS1) to form acyl-Co esters. 5: VLC-FA are preferentially transported by FATP1 (or other FATPs; see text) and by action of the synthetase activity of FATP1 directly converted into VLC-acyl-CoA esters (uptake by vectorial acylation). B: schematic presentation of the proposed topology of CD36 (two transmembrane domains), with the small cytoplasmic tails palmitoylated. The large extracellular loop has 10 putative N-linked glycosylation sites and two phosphorylation sites. Disulfide bonds between the extracellular cysteines are also shown (between amino acid residues 243–311, 272–333, and 313–323). The NH2 and COOH termini each contain two palmitoylation sites, and the COOH terminus contains two ubiquitination sites. The shaded area designates a hydrophobic pocket comprised by amino acid residues 93–183 that likely is involved in ligand binding (residues 93–120 was identified as the thrombospondin binding site, residues 120–155 was mapped for oxidized LDL, and residues 139–183 form a multiligand binding site). Arrowheads and numbers indicate the approximate positions of amino acid residues. [Data compiled from Tao et al. (427), Febbraio and Silverstein (125), Ibrahimi et al. (214), and Hoosdally et al. (205).] C: proposed topology of FATP1. Only a short segment of the NH2 terminus faces the extracellular side of the membrane. Amino acid residues 1–190 are integrally associated with the membrane, while residues 190 –257 are cytosolic and contain the AMP-binding motif that mediates the acyl-CoA synthetase activity. Amino acid residues 258 – 475 are peripherally associated with the inner leaflet of the cell membrane. The COOH terminus is located in the cytoplasm. Arrowheads and numbers indicate the approximate positions of amino acid residues. [Data compiled from Lewis et al. (266) and DiRusso et al. (103).]

FIG. 4. Similarity between the regulation of cellular uptake of fatty acids and glucose. The uptake of both fatty acids and glucose by cardiac and skeletal muscle is increased after translocation of specific transporter proteins (shown for CD36 and GLUT4, respectively) to the sarcolemma in response to stimulation with insulin or during increased contractile activity. CD36 and GLUT4 may be mobilized from different stores within the endosomal compartment. Note that, for clarity, the involvement of GLUT1 in glucose uptake and the recycling of other fatty acid transporters (FABPpm and FATPs) are not shown, while recently it has been observed that they can also be induced to translocate, at least in skeletal muscle (219). FA, (long-chain) fatty acid.

FIG. 5. Trafficking pathways involved in CD36 recycling. The transition of CD36 from the endosomal storage compartment to the plasma membrane, and vice versa, is regulated by several protein families with different functions. 1, Bottom inset: adaptor proteins and Rab-GTPases regulate the binding of coat proteins to areas enriched in CD36. The formation of coat complexes leads to the generation of a CD36 transport vesicle which is released to the cytoplasm by scission proteins. After budding of the vesicle, the adaptor and coat proteins again detach. 2: Upon binding of a motor protein, the transport vesicle is moved along cytoskeleton filaments to its designated target membrane. Also this process is initiated by Rab-GTPases. 3, Top inset: tethering proteins trap the transport vesicle below the cell surface. The interaction of a specific subset of vesicleassociated SNAREs (v-SNAREs) and target-membrane associated SNAREs (t-SNAREs) brings the vesicle to such a close proximity to the plasma membrane that the water barrier can be overridden and fusion of the two membranes can occur. After fusion, the SNARE complex disintegrates. 4–6: The transport of CD36 from the plasma membrane to the endosomal compartment proceeds analogous to steps 1–3.

FIG. 7. Changes in substrate utilization in hearts from CD36 null mice. Rates of palmitate oxidation (left), glucose oxidation (middle), and tricarboxylic acid (TCA) cycle acetyl-CoA production (right) were determined in wild-type and CD36 null mouse hearts perfused as working hearts with 1.2 mM palmitate and 5 mM glucose. TCA cycle activity was calculated from the rates of palmitate and glucose oxidation, using 8 mol acetyl-CoA for every 1 mol palmitate oxidized and 2 mol acetylCoA for every 1 mol glucose oxidized. The lower palmitate oxidation seen in hearts from CD36 null mice is compensated by an increase in glucose oxidation so that these hearts are not energetically compromised. [Redrawn from Kuang et al. (258), with permission from the American Heart Association.]

FIG. 2. Characterization of fatty acid transport into giant sarcolemmal vesicles obtained from heart and skeletal muscle. A: kinetics of palmitate transport into giant sarcolemmal vesicles from rat heart and red and white skeletal muscle. B: correlation between the rate of palmitate transport into giant sarcolemmal vesicles and the amount of the fatty acid transporter CD36 located on the plasma membrane of these vesicles. C: correlation between the rate of palmitate transport into giant sarcolemmal vesicles and the fatty acid oxidation capacities of the tissues. [Redrawn from studies by Bonen et al. (45), Glatz et al. (144), and Luiken et al. (286).]

FIG. 6. Chronically altered muscle contractile activity affects the rate of fatty acid uptake and plasmalemmal CD36 and FABPpm. Rat hindlimb muscles were chronically stimulated (7 days) or denervated (7 days) whereafter fatty acid transport into giant sarcolemmal vesicles and plasmalemmal CD36 (left) and FABPpm (right) content were determined. FATPs were not studied. [Redrawn from Koonen et al. (252).]

Full text

Membrane Fatty Acid Transporters as Regulators of
Lipid Metabolism: Implications for Metabolic Disease
Citation for published version (APA):
Glatz, J. F. C., Luiken, J. J. F. P., & Bonen, A. (2010). Membrane Fatty Acid Transporters as Regulators
of Lipid Metabolism: Implications for Metabolic Disease. Physiological Reviews, 90(1), 367-417.
https://doi.org/10.1152/physrev.00003.2009
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Membrane Fatty Acid Transporters as Regulators of Lipid
Metabolism: Implications for Metabolic Disease
JAN F. C. GLATZ, JOOST J. F. P. LUIKEN, AND AREND BONEN
Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands;
and Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Canada
I. Introduction 368
II. Mechanism of Transmembrane Transport of Fatty Acids 368
A. Membrane fatty acid transport mediated by lipids or proteins? 369
B. Evidence for the involvement of membrane proteins 370
III. Membrane-Associated Fatty Acid Transporters 373
A. Plasma membrane fatty acid binding protein 373
B. Fatty acid translocase/CD36 374
C. Fatty acid transport proteins 375
D. Caveolins 377
E. Overall conclusions on fatty acid transporters 378
IV. Functioning and Subcellular Localization of Fatty Acid Transporters 378
A. Subcellular translocation of fatty acid transporters 378
B. Posttranslational modification of fatty acid transporters 381
C. Functioning of fatty acid transporters in mitochondrial fatty acid utilization 382
D. Coordinated functioning of fatty acid transporters 383
E. Do fatty acid transporters channel fatty acids to a particular metabolic fate? 384
V. Signaling and Trafficking Events Regulating Membrane Transporter Translocation 385
A. Signaling pathways 385
B. Trafficking pathways 389
VI. Chronic Physiological Regulation of Fatty Acid Transporters 391
A. Regulation of fatty acid transporter expression 391
B. Effects of development, ageing, and gender 392
C. Effects of fasting, hormones, and exercise training 393
VII. Alterations in Fatty Acid Transporters in Disease 394
A. Cardiac hypoxic disease and heart failure 394
B. Insulin resistance and type 2 diabetes 396
C. Type 1 diabetes 402
VIII. Conclusions and Perspectives 403
A. Integration of regulatory steps 403
B. Fatty acid transporters as potential therapeutic targets 404
Glatz JFC, Luiken JJFP, Bonen A. Membrane Fatty Acid Transporters as Regulators of Lipid Metabolism:
Implications for Metabolic Disease. Physiol Rev 90: 367–417, 2010; doi:10.1152/physrev.00003.2009.—Long-chain fatty
acids and lipids serve a wide variety of functions in mammalian homeostasis, particularly in the formation and dynamic
properties of biological membranes and as fuels for energy production in tissues such as heart and skeletal muscle. On
the other hand, long-chain fatty acid metabolites may exert toxic effects on cellular functions and cause cell injury.
Therefore, fatty acid uptake into the cell and intracellular handling need to be carefully controlled. In the last few years,
our knowledge of the regulation of cellular fatty acid uptake has dramatically increased. Notably, fatty acid uptake was
found to occur by a mechanism that resembles that of cellular glucose uptake. Thus, following an acute stimulus,
particularly insulin or muscle contraction, specific fatty acid transporters translocate from intracellular stores to the
plasma membrane to facilitate fatty acid uptake, just as these same stimuli recruit glucose transporters to increase
glucose uptake. This regulatory mechanism is important to clear lipids from the circulation postprandially and to rapidly
facilitate substrate provision when the metabolic demands of heart and muscle are increased by contractile activity.
Studies in both humans and animal models have implicated fatty acid transporters in the pathogenesis of diseases such
as the progression of obesity to insulin resistance and type 2 diabetes. As a result, membrane fatty acid transporters are
now being regarded as a promising therapeutic target to redirect lipid fluxes in the body in an organ-specific fashion.
Physiol Rev 90: 367– 417, 2010;
doi:10.1152/physrev.00003.2009.
www.prv.org 3670031-9333/10 $18.00 Copyright © 2010 the American Physiological Society
Downloaded from journals.physiology.org/journal/physrev at Universiteit Maastricht (137.120.149.252) on September 27, 2021.

I. INTRODUCTION
The importance of long-chain fatty acids and lipids
for mammalian homeostasis is well recognized. Fatty ac-
ids (for convenience this term is used to designate “long-
chain fatty acids,” unless otherwise indicated) are primar-
ily known as constituents of “fat,” which represents a
crucial and efficient energy store due to the high energy
content per unit weight. Apart from their fundamental
role as a fuel for energy production, fatty acids are incor-
porated into phospholipids forming the core of biological
membranes and serve in selected signal transduction
pathways to alter gene expression. However, largely due
to their hydrophobic properties, fatty acids also exert
harmful effects and may cause (acute) cellular injury (96,
235, 468). Taken together, these divergent characteristics
of fatty acids require that their transport among and into
tissues occurs through specific mechanisms that allow
their rapid and controlled distribution without the possi-
ble detrimental effects associated with their detergent-
like properties.
Dietary fats typically comprise 30–40% of energy
intake and consist mostly of long-chain fatty acids ester-
ified in triacylglycerols. Lingual and pancreatic lipases
will hydrolyze these triacylglycerols into monoacylglyc-
erol and fatty acids which then are taken up by jejunal and ileal
enterocytes, reesterified into triacylglycerols, and incor-
porated with other lipids, lipid-soluble vitamins, and apo-
lipoproteins into chylomicrons for subsequent secretion
into the circulation. Similarly, the liver secretes very-low-
density lipoproteins produced from fatty acids synthe-
sized de novo or taken up from blood plasma and subse-
quently esterified into triacylglycerols and apolipopro-
teins. Both chylomicrons, carrying exogenous lipids, and
very-low-density lipoproteins, carrying endogenous lipids,
undergo hydrolysis of their triacylglycerols by lipoprotein
lipase located at the surface of the capillaries, so as to
deliver the fatty acids into peripheral tissues. Fatty acids
stored in adipocytes are hydrolyzed by hormone-sensitive
lipase (HSL) and adipose tissue triacylglycerol lipase
(ATGL), and distributed to other tissues bound to albumin
via the circulation. Taken together, a complex system
operates to distribute fatty acids among various tissues.
The uptake of fatty acids by parenchymal cells, es-
pecially their translocation across the cell membrane, has
long been considered to occur by simple (passive) diffu-
sion, with the rate of uptake being determined primarily
by the rate of fatty acid delivery (blood flow ⫻ extracel-
lular concentration) and the rate of intracellular fatty acid
metabolism. However, from a physiological perspective, it
would be highly desirable to regulate the entry of fatty
acids into the cell to tune their uptake to the metabolic
needs and avoid possible harmful effects of excess fatty
acid accumulation. Specifically, the objective of such con-
trol would be 1) to ensure fatty acid uptake when its
extracellular concentration is relatively low, 2) to limit
uptake when the extracellular fatty acid concentration is
relatively high, 3) potentially select for specific fatty acid
types, and 4) allow rapid adjustments in fatty acid provi-
sion at the local tissue level to meet rapid fluctuations in
metabolic demands, especially in heart and skeletal mus-
cle.
In the past few decades it has become clear that
various membrane-associated fatty acid-binding proteins
(termed “fatty acid transporters,” for convenience) facil-
itate the cellular entry of fatty acids, which are then
accepted by cytoplasmic fatty acid binding proteins
(FABP
c
). Furthermore, it has been found that acute
changes in fatty acid uptake in response to mechanical
(e.g., muscle contraction) and hormonal stimuli (insulin)
are regulated by specific membrane proteins, in a fashion
similar to the regulation of glucose uptake by glucose
transporters. Finally, studies in both humans and animal
models have implicated the membrane fatty acid trans-
porters in various metabolic aberrations and pathologies.
Thus a selective expression and/or regulation of specific
(sets of) membrane-associated and cytoplasmic fatty acid-
binding proteins could contribute to the control of the
fatty acid uptake and utilization processes, thereby en-
abling tissue-specific fatty acid uptake and utilization in-
dependent of fatty acid delivery. However, while FABP
c
inside the cell functions as a sink for incoming fatty acids,
it displays merely a permissive action in cellular fatty acid
uptake in that only its full ablation reduces the rate of
cellular fatty acid uptake and utilization (32, 33, 280, 370;
for detailed reviews of FABP
c
, see Refs. 142, 188, 407). In
contrast, it appears that specific (sets of) plasma mem-
brane-associated proteins are central to regulating fatty
acid uptake and utilization.
In this review we discuss our current understanding
of the role of membrane fatty acid transporters in cellular
lipid metabolism, focusing on both the acute and chronic
regulation of cellular fatty acid uptake and on chronic
metabolic diseases, including myocardial disease, insulin
resistance, and types 1 and 2 diabetes. Data are presented
mostly for heart and skeletal muscle, as these tissues have
been studied most intensively, but the concepts to be
outlined generally may also apply to other tissues (see
sect. VIII). Other related and recent reviews have ad-
dressed changes in lipid and carbohydrate metabolism in
the failing heart (400), fatty acid metabolism in the type 2
diabetic heart (66), and skeletal muscle lipid metabolism
in exercise and insulin resistance (242).
II. MECHANISM OF TRANSMEMBRANE
TRANSPORT OF FATTY ACIDS
In recent years there has been considerable debate
on the mechanism by which fatty acids are taken up by
368 GLATZ, LUIKEN, AND BONEN
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cells, that is, how fatty acid transport occurs across the
plasma membrane, between the aqueous phases on either
side of this barrier. The dispute centers around the rate-
limiting kinetic step in this process, being either the ad-
sorption of fatty acids to, or insertion into, the outer
leaflet of the lipid bilayer, the subsequent transfer to the
inner leaflet (referred to as flip-flop), or the desorption
from the membrane into the aqueous phase (163), and
whether one or more membrane proteins could facilitate
either one or all of these steps or serve distinct functions
in the overall uptake process. Below we discuss specific
features and limitations of the methodologies and the
experimental models used. The reader is referred to early
(161, 163) and more recent (162, 233) reviews of the
controversies concerning the rate-limiting processes in-
volved in transferring fatty acids through the plasma
membrane.
A. Membrane Fatty Acid Transport Mediated
by Lipids or Proteins?
When considering the cellular uptake of fatty acids, the
physical transport can be regarded as seven kinetic steps:
1) dissociation of fatty acid from extracellular albumin into
the aqueous phase; 2) diffusion through the outer aqueous
phase; 3) insertion into the outer leaflet of the phospholipid
bilayer; 4) flip-flop from the outer to the inner leaflet, defined
as the complete movement of the fatty acid across the
bilayer with reorientation of the carboxyl head group from
the outer lipid-water interface to the inner lipid-water inter-
face; 5) dissociation from the inner leaflet; 6) diffusion
through the inner aqueous phase; and 7) binding to FABP
c
.
Thereafter, the fatty acid may be activated to its acyl-CoA
ester and undergo further metabolism.
The aqueous solubility of fatty acids, earlier esti-
mated to be in the micromolar range (395), is now recog-
nized to be extremely low, in the range 1–10 nM (465),
indicating that virtually all of the fatty acids will be
present in membranes or bound to proteins. The soluble
fatty acid binding proteins allow fatty acids to be miscible
in aqueous environments. Thus albumin in the circulation
and interstitium (348) and FABP
c
in the cytoplasm (141,
350) act as extracellular and intracellular buffers, respec-
tively, for fatty acids so that under normal physiological
conditions (total fatty acid concentration in the range
100–400
␮
M) generally only ⬍1 part in 10
5
is present in
the aqueous phase. In line with this, the average concen-
tration of (non-protein bound) fatty acids in human
plasma was reported to be 7.5 ⫾ 2.5 nM (349).
Different approaches and model systems have been
used to delineate the rate governing kinetic step in the
overall cellular fatty acid uptake process. Various groups
have studied fatty acid transport across the lipid bilayer of
artificial phospholipid vesicles by incubating these vesi-
cles with fatty acids, or albumin-fatty acid complexes, and
monitoring either the appearance of fatty acids in the
internal aqueous phase of the vesicle or the change in pH
inside the vesicle that occurs as a result of the transmem-
brane movement of fatty acids. The intravesicular fatty
acid concentration has been measured using ADIFAB, a
fluorescent probe composed of acrylodan-derivatized in-
testinal type FABP
c
that allows the accurate assessment
of very low concentrations (nM) of fatty acids in aqueous
solutions without disturbing their binding equilibrium
with proteins or membranes (351). ADIFAB has been
trapped into phospholipid vesicles or erythrocyte ghosts
during their formation and has also been microinjected
into adipocytes (234). Alternatively, a pH-sensitive fluoro-
phore such as pyranine or 2⬘,7⬘-bis(2-carboxyethyl)-5(6)-
carboxyfluorescein (BCECF), has been trapped inside
phospholipid vesicles, to monitor the H
⫹
that dissociates
from the transported (un-ionized) fatty acid upon its ap-
pearance at the inner leaflet of the bilayer (see below) (84;
for review, see Ref. 59). In earlier studies transport has
also been measured with fluorescently labeled fatty acid
analogs (251, 408), but the addition of a large fluorescent
moiety is expected to dramatically alter the physicochem-
ical properties of fatty acids, and therefore alter transport
rates (233). Because of these considerations, these stud-
ies will not be discussed here.
Hamilton and co-workers have monitored the move-
ment of fatty acids across phospholipid membranes using
pH-sensitive probes. When presented either as albumin-
fatty acid complex, dissolved in organic solvent, or as the
K
⫹
soap, the fatty acids rapidly partition into the outer
leaflet of the membrane. Because in such environment the
apparent pK
a
of the fatty acid shifts from ⬃4.5 in an
aqueous solution to ⬃7.6 (independent of fatty acid type),
about half of the fatty acids are present in the un-ionized,
i.e., protonated, form. This uncharged species can then
easily flip-flop without electrochemical restrictions to the
inner leaflet of the membrane, after which a proton is
donated to the interior solution and the fatty acid is
available for desorption (229). Applying this approach to
studies with phospholipid vesicles (230) and with adipo-
cytes (84, 228) revealed linear relationships between the
quantity of added fatty acids, the amount of fatty acids
that binds to the plasma membrane, and the decrease in
intracellular pH. From these various studies it was con-
cluded that binding of the fatty acid to the membrane
(adsorption) occurs extremely fast, seems to be diffusion-
limited, and is largely independent on the fatty acid chain
length, and that transbilayer movement is fast (t
1/2
⬍1s)
for all fatty acid types and fast in cells (⬃⬍10 s). These
observations have been interpreted to infer that fatty
desorption from the membrane may be the rate-limiting
step for the overall transport rate of fatty acids, at least
for a protein-free model membrane (which represents an
FATTY ACID TRANSPORTERS AND LIPID METABOLISM 369
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artificial situation). A more recent study has provided
further evidence for this concept (383).
Kleinfeld and colleagues (97) used similar model sys-
tems to study transmembrane fatty acid transport, apply-
ing ADIFAB to detect fatty acid influx, to observe virtually
identical overall rates of transport as reported by Hamil-
ton’s group (228). However, in contrast to Hamilton, they
concluded that flip-flop is rate-limiting, since their data
showed that the dissociation of fatty acids from the mem-
brane is faster than flip-flop (97, 250). The discrepancies
with other reports have been attributed to 1) the absence
of albumin in some of these other studies which exposes
the membranes to high (⬎5
␮
M) concentrations of fatty
acids that perturb the bilayer structure, and 2) to misin-
terpretation of the measurements (233). More recently,
Kampf and Kleinfeld (232) have used quantitative fluores-
cence ratio microscopy to measure (noninvasively) fatty
acid transport into adipocytes by imaging the intracel-
lular (non-protein bound) fatty acid concentrations
(232). Their results indicate that transport rate con-
stants are ⬎50-fold slower in adipocytes than in artifi-
cial phospholipid vesicles that contain no proteins,
such as are normally present in biological membranes.
From these data they conclude that fatty acid transport
across adipocyte membranes is highly regulated and
best described by a membrane carrier model (for re-
view, see Ref. 233).
In summary, studies in protein-free artificial mem-
branes show that passive flip-flop of the un-ionized form
of fatty acids can occur rapidly and in a protein-indepen-
dent manner across the lipid bilayer phase, indicating that
the lipid bilayer does not represent a barrier for fatty
acids (Fig. 1A). However, contrary to most findings with
these protein-deficient, synthetic lipid vesicles, newer
studies with cellular preparations that contain proteins
and which apply noninvasive techniques to monitor fatty
acid uptake, suggest that flip-flop is the rate-limiting step
for fatty acid transport across lipid bilayers (233, 234).
Because flip-flop is relatively slow and dependent on the
membrane structure (being slower through the ordered
phase than through the liquid-crystalline phase), diffusion
rates through the lipid bilayer may not be sufficiently
rapid to meet the metabolic demands of certain cells
and/or under certain conditions, particularly cells in
which the metabolic demands for fatty acids can be rap-
idly upregulated (e.g., heart and skeletal muscle). This
implies that at least certain biological tissues may require
membrane proteins to catalyze the flip-flop step (231).
Such proteins could act as transmembrane transporters
for fatty acids, but they could also attract albumin or
other fatty acid carriers and enhance the concentration of
fatty acids near the membrane surface, which would help
overcome the barriers of the unstirred water layer. Another
possibility is that membrane proteins act as a sink for fatty
acids, as has been proposed for caveolin-1 which has mul-
tiple basic residues at its intracellular domain that could
interact with the carboxylate anion and in this way acceler-
ate transmembrane fatty acid transport (see sect. IIID) (299).
A prevalent view is that both passive diffusion and
protein-mediated transport contribute to the cellular
uptake of fatty acids. Estimates of the contributions of
these two mechanisms have been made by deconvolu-
tion of uptake curves and by the use of inhibitors of
protein-mediated uptake. Because of saturation of the
protein-mediated component at high fatty acid concen-
tration, most of these studies have been interpreted to
suggest that protein-mediated uptake is important at
physiological concentrations of fatty acids and that
passive diffusion becomes predominant at higher, pre-
sumably nonphysiological concentrations of fatty acids
(2, 3). However, others feel that such data need to be
interpreted with caution (161). Still others have ques-
tioned the coexistence of diffusional and protein-mediated
fatty acid transport across the membrane’s lipid phase and
have proposed that fatty acid movement across the plasma
membrane is primarily protein mediated (232, 233).
Taken together, the unifying concept arises that
during the process of cellular uptake, fatty acids rapidly
bind and partition into the plasma membrane, then may
undergo lateral diffusion to specific domains such as
lipid rafts (333) before their desorption into the intra-
cellular compartment. Membrane proteins thus would
function in regulating fatty acid entry into the cell by
1) adsorbing fatty acids from the extracellular media
and modulating their transport into the membrane, and
2) segregating or organizing fatty acids for metabolism.
B. Evidence for the Involvement of
Membrane Proteins
Starting in the early 1980s, investigators from dif-
ferent laboratories reported that the uptake of fatty
acids into various parenchymal cell types showed
1) saturation kinetics, 2) sensitivity to general inhibi-
tors of protein-mediated plasma membrane transport
processes (e.g., phloretin and proteases), 3) sensitivity
to inhibition by nucleophilic fatty acid derivatives (e.g.,
sulfo-N-succinimidyloleate, SSO; later shown to specif-
ically inhibit CD36, see sect. IVA1), and 4) sensitivity to
competitive inhibition (5, 6, 287, 412). Although those
observations each are in favor of protein-mediated
transport, they have been disputed by others (161, 162,
358) who argued that saturation of fatty acid transport
can also be explained as saturation of metabolism in
combination with passive diffusion. Moreover, the used
inhibitors could theoretically inhibit fatty acid uptake via
indirect effects on the structural organization of the bilayer,
and the fatty acid competition experiments could unveil
competition for albumin rather than for transporters.
370 GLATZ, LUIKEN, AND BONEN
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Frequently asked questions

Q1. What have the authors contributed in "Membrane fatty acid transporters as regulators of lipid metabolism: implications for metabolic disease" ?

• A submitted manuscript is the version of the article upon submission and before peer-review. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher 's website. The final author version and the galley proof are versions of the publication after peer review. The final published version features the final layout of the paper including the volume, issue and page numbers. 

Q2. Why are giant vesicles used to study fatty acid uptake?

Because of the absence of subcellular organelles or metabolic enzymes, giant vesicles can be used to study substrate uptake dissected from metabolism. 

Q3. What are the defining protein constituents of caveolae?

Caveolins are the defining protein constituents of caveolae, which are specialized microdomains of the plasma membrane, enriched in cholesterol, sphingomyelins, and signaling and receptor proteins (88, 327). 

Q4. What is the driving gradient for net fatty acid movement in heart and muscle?

For instance, in heart and muscle, the driving gradient for net fatty acid movement is always from the extracellular space into the myocyte, while in adipose tissue fatty acid transport may be directed into or out of adipocytes. 

Q5. What could be the role of membrane proteins in fatty acid transport?

Such proteins could act as transmembrane transporters for fatty acids, but they could also attract albumin or other fatty acid carriers and enhance the concentration of fatty acids near the membrane surface, which would help overcome the barriers of the unstirred water layer. 

Q6. How does FATP1 affect long-chain fatty acid transport?

-2, and -4 are particularly effective in facilitating the rates of long-chain fatty acid transport by 8.2-, 4.5-, and 13.1-fold, respectively, whereas FATP3 and -5 provide only a modest 2-fold increase, and FATP6 provides virtually no increase in long-chain fatty acid transport (104). 

Q7. How did knockdown studies of FATP1 affect basal fatty acid uptake?

Knockdown studies of FATP1 (3T3-L1 adipocytes) revealed that basal fatty acid uptake was reduced (268), whereas with FATP4 knockdown (3T3-L1 adipocytes) or overexpression (HEK-293 cells), there were no changes in fatty acid uptake (268). 

Q8. What is the role of caveolae in CD36?

CD36 is associated with the cholesterol- and sphingolipid-rich membrane microdomains known as rafts (or as caveolae when they contain caveolin). 

Q9. What is the effect of blocking ACS1 on fatty acid uptake?

Overexpression of each of these two proteins increases fatty acid uptake, and their concomitant overexpression has a synergistic effect on fatty acid uptake (136), while blocking ACS1 activity reduces fatty acid uptake (346). 

Q10. What is the effect of the addition of a large fluorescent moiety on transport?

In earlier studies transport has also been measured with fluorescently labeled fatty acid analogs (251, 408), but the addition of a large fluorescent moiety is expected to dramatically alter the physicochemical properties of fatty acids, and therefore alter transport rates (233). 

Q11. What is the role of caveolins in the plasma membrane?

Caveolins are responsible for the invagination of the plasma membrane, giving the caveolar microdomains their flask-shaped appearance. 

Q12. What is the role of caveolae in fatty acid uptake?

One remarkable issue is that all fatty acid transporters also appear to have functions that are unrelated to fatty acid transport; for example, FABPpm and the FATPs contain mAspAt activity and VLACS activity, respectively; CD36 displays multiple other functions, including thrombospondin binding; caveolins possess the ability to form caveolar regions. 

Q13. What is the reason for the discrepancies with other studies?

The discrepancies with other reports have been attributed to 1) the absence of albumin in some of these other studies which exposes the membranes to high ( 5 M) concentrations of fatty acids that perturb the bilayer structure, and 2) to misinterpretation of the measurements (233). 

Q14. What is the role of membrane proteins in regulating fatty acid transport?

Membrane proteins thus would function in regulating fatty acid entry into the cell by 1) adsorbing fatty acids from the extracellular media and modulating their transport into the membrane, and 2) segregating or organizing fatty acids for metabolism. 

Q15. What is the dispute about the ratelimiting kinetic step in the transport of fatty acids?

The dispute centers around the ratelimiting kinetic step in this process, being either the adsorption of fatty acids to, or insertion into, the outer leaflet of the lipid bilayer, the subsequent transfer to the inner leaflet (referred to as flip-flop), or the desorption from the membrane into the aqueous phase (163), and whether one or more membrane proteins could facilitate either one or all of these steps or serve distinct functions in the overall uptake process.