The Cellular Fatty Acid Binding Proteins: Aspects of Structure, Regulation, and Function

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. III

The Cellular Fatty Acid Binding Proteins: Aspects of Structure, Regulation, and Function NATHAN M. BASS Department of Medicine and Liver Center, University of California, San Francisco, California 94143

I. Introduction

Long chain fatty acids provide the main energy source of most mammalian tissues and also comprise essential components of the structural lipids of cell membranes. The considerable daily flux of long chain fatty acids released into the plasma from adipose tissue triglyceride stores (about 700 mmol/day in the average human), as well as their poor solubility in water and potential for forming injurious detergent micelles, are managed and offset by a complex set of resources within the organism. These include the storage of fatty acids in tissues as well as their transport in plasma lipoproteins largely in the form of relatively inert triglycerides, and the abundant presence of fatty acid binding proteins, with high affinities for unesterified long chain fatty acids, in both the extracellular and intracellular aqueous compartments. Serum albumin serves as the main fatty acid binding protein in the plasma. Recent evidence has suggested that the role of albumin is not confined t o that of a mere “shuttle” for fatty acids and other ligands between their sites of entry and removal from the plasma, but that it may also in part determine the sites of removal of its ligands (Okajima et al., 1985; Inoue et al., 1986), and may regulate the hepatic removal of bound fatty acids and other small molecules in a complex and poorly understood manner (Forker and Luxon, 1981; Weisiger et al., 1981; Ockner et al., 1983: Weisiger, 1985; Mizuma et al., 1985; Oie and Fiori, 1985; Fleischer et al., 1985, 1986; Tsao et al., 1986). The existence of an abundant fatty acid binding protein present in the Mr 12,000 fraction of the cytosol of several mammalian tissues was first reported by Ockner et al. (1972a) and Mishkin el GI. (1972). Over the subsequent 14 years, the intracellular fatty acid binding proteins (FABP) have been extensively studied in relation t o their structure, function, and regulation. In many respects, these proteins appear to represent the intracellular equivalent to serum albumin, participating in the intracellular storage and transport of fatty acids, and influencing their metabolic I43

Copyright 8 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.

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utilization via as yet poorly understood mechanisms. Several recent reviews have covered a variety of aspects of the purification, quantitation, structure, regulation, and function of the FABP (Glatz and Veerkamp, 1985; Bass, 1985; Glatz et al., 1985a; Gordon and Lowe, 1985; Haq and Shrago, 1985; Ockner, 1986). The aim of the present article is to provide a comprehensive overview of our current state of knowledge of this expanding field of research with particular emphasis on evolving concepts of FABP structure, regulation, and function.

11. The Cellular FABP

A. CYTOSOLIC FABP Early studies employed gel filtration binding assays of radiolabeled fatty acids and other ligands to identify and quantitate FABP in tissue cytosol fractions (Ockner et al., 1972a; Mishkin et al., 1972). Purification of the specific proteins responsible for the low-molecular-weightfatty acid binding protein activity in different tissues has led to the characterization of three structurally distinct FABP of similar size, each the product of a separate gene. The three cytosolic FABP are generally named according to their tissues of greatest abundance, and comprise liver FABP (Ockner et al., 1982; Trulzsch and Arias, 1981b), intestinal FABP (Ockner and Manning, 19741, and heart muscle FABP (Fournier et al., 1978; Said and Schultz, 1984).Table I lists the synonyms and comparative physicochemical properties of the cellular FABP in the rat, the species in which these proteins have been most extensively characterized. Preliminary evidence has also been presented for the existence of a fourth molecular type of 12- to 14-KDa FABP in rat skeletal muscle (Said and Schultz, 1985). FABP identified in the tissues of other mammalian and vertebrate species, including human (Kamisaka et d., 1981; Unterberg et d . , 1986), bovine (Haunerland et al., 1984; Jagshies et al., 1985; Whetstone et al., 1986), pig (Fournier and Rahim, 1983), rabbit (Matsushita et al., 1977; Vincent et al., 1985), mouse, guinea pig (Kawashima et al., 1984), and avian species (Katongole and March, 1979; Lee and Wiggert, 1984), exhibit largely similar physicochemical and binding properties to the rat proteins. A M,12,000 protein with organic anion binding properties similar to those of mammalian FABP has been found in the livers of marine elasmobranchs (Sugiyama et al., 1982a), but it curiously showed no binding activity with fatty acids. A 14.6-kDa protein structurally related to myelin P, protein and the rat cytosolic FABP is abundantly expressed during the differentiation of mouse 3T3-L 1 preadipocytes to mature adipocytes (mouse

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TABLE 1 COMPARATIVE PROPERTIES OF THE CELLULAR FABP IN THE RAT Protein

Synonymdabbreviation

Liver FABP

L-FABP hepatic FABP (h-FABP) Z-protein' Azocarcinogen binding protein Ad Heme binding protein (HBP)' Sterol carrier protein (SCP)' Intestinal FABP I-FABP gut FABP (g-FABP) Heart muscle FABP M-FABP Liver plasma LPM-FABP membrane FABP

Size (Da)

PI

Main tissues

14,184"

3-5 isoforms pl 5.2-6.9'

Liver, small intestine

15,0638

5.6h

Small intestine

14,992' 4O,OOOL

5.w 9.0"

Heart, skeletal muscle Liver, small intestine, heart muscle

"Gorden et a / . (1983). hTrulzsch and Arias (1981b): Ockner C I a / . (1982). 'Levi e: a / . (1%9): Trulzsch and Arias (1981b). "Ketterer e: a/. (1%7). "Vincent and Muller-Eberhard (1985). 'Dempsey e: a / . (1981. 1985). #Alpers e: a / . (1984). "Ockner and Manning (1974). 'Sacchettini e: a / . (1986). 'Fournier el a / . (1978). 'Stremmel er a / . (1985b).

adipocyte p422 protein) (Bernlohr el al., 1984). Available data suggest that the p422 protein may function as an FABP in mouse adipocytes (Ockner ef af., 1982). An 8.7-kDa protein with a high affinity for long chain fatty acids has been purified from oat seedlings (Rickers et al., 1984), but its phylogenetic relationship to the mammalian FABP is unknown. B. OTHERCELLULAR FABPs A poorly characterized 1.5-kDa FABP has been described in rat liver that is not a degradation product of the larger FABP, and which may be a component of microsomes and mitochondria (Suzue and Marcel, 1975; Rustow et af., 1979). The major 71- to 73-kDa heat-shock proteins of rat liver and brain have also been found to contain tightly bound fatty acids in a ratio of 4 mol fatty acid/mol of protein dimer (Guidon and Hightower, 1986a,b). A most significant recent development has been the purification

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of a 40-kDa FABP from the plasma membrane of liver and intestinal brush border by Stremmel and co-workers (1985a,b). The putative function of this protein in the translocation of fatty acids across the cell membrane is also discussed in this review (Section VI1,C). C. NOMENCLATURE Liver FABP (L-FABP, h-FABP) has accumulated several synonyms (Table I) bestowed by different groups who have isolated and studied this protein in relation to its properties as a bilirubidorganic anion binding protein (Z-protein; Levi et al., 1969), a carcinogen binding protein (azocarcinogen binding protein A; Ketterer et al., 1967),a heme binding protein (HBP, Vincent and Muller-Eberhard, 1985), and a sterol and squalene carrier protein (SCP, Dempsey, 1975, 1984; Dempsey et al., 1985). The situation is much simpler regarding intestinal FABP, which has been referred to in abbreviated form as either I-FABP or g(for gut)-FABP, and heart muscle FABP, which has no suggested abbreviated form as yet. For the purpose of this review, the following abbreviated nomenclature will be used: L-FABP for cytosolic liver FABP, I-FABP for cytosolic intestinal FABP, and M-FABP for cytosolic heart muscle FABP. LPM-FABP will refer to the 40-kDa FABP isolated from liver plasma membranes (Stremmel et al., 1985b). One major source of confusion is also worth pointing out at this juncture. The protein isolated by Dempsey and co-workers and designated SCP (sterol and squalene carrier protein) appears to be identical with FABP on the basis of hepatic abundance, immunological identity, and even amino acid sequence (Dempsey et al., 1985; Chan et al., 1985). Nevertheless, SCP has been found by Dempsey er af. (1985) to exhibit several aspects of regulation and tissue expression quite different from the findings reported by others for L-FABP (e.g., see Sections IV and V1,B). One possible explanation for this phenomenon is that SCP preparations may contain additional proteins quite distinct from L-FABP. Thus, in some instances, due regard is given to this possibility by referring to SCP/LFABP when quoting certain studies. It is also important to note that the SCP described by Dempsey et af. (1985) is quite different from the two cytosolic sterol carrier proteins designated SCP, and SCP2. The former is a 47-kDa protein that participates in the microsomal conversion of squalene to lanosterol (Scallen et al., 1974, 1985b). The latter is a 13.5kDa protein that participates in the microsomal conversion of lanosterol to cholesterol (Scallen et al., 1985b),which is identical to liver nonspecific lipid transfer protein (nsL-TP), and is quantitatively, functionally, and structurally quite different from L-FABP (Van Amerongen et af., 1985; Westerman and Wirtz, 1985).

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111. FABP Structure

Complete primary sequence data have been obtained by peptide and cDNA nucleotide sequencing for L-FABP in the rat (Takahashi et al., 1982a,b, 1983; Gordon et al., 1983) and human (Chan et al., 1985; Lowe et al., 1985), and for rat I-FABP (Alpers et al., 1984), and M-FABP (Sacchettini et al., 1986). The amino acid sequences for the three rat cytoplasmic FABPs are shown in Fig. 1. In keeping with their characterization as nonsecreted cytosolic proteins, all three FABPs have blocked (acetylated) N-terminal residues (Gordon et al., 1983; Alpers et al., 1984; Sacchettini et al., 1986), and, in addition, the primary translation products of L-FABP and I-FABP contain no cleavable signal peptide sequences (Gordon and Lowe, 1985). L-FABP is not glycosylated (Haunerland et al., 1984) and although evidence for covalent attachment of fatty acids to this protein has been presented (Dempsey et al., 1981), others have been unable to confirm this (Haunerland et al., 1984). Covalent attachment of fatty acids to FABP purified from bovine mammary gland has also been reported (Whetstone et al. 1986). Predictions of the secondary structure of L-FABP based upon sequence data have assigned it between 29 and 40% a helix (Takahashi et al., 1983; Gordon et al., 1983; Chan et al., 1985), which is somewhat greater than the 14% a-helical structure found on circular dichroism (CD) studies (Kamisaka et al., 1975). CD spectra of M-FABP predict between 15-22% a helix, 45-51% p form, and 2633% random form for this protein (Jagschies et al., 1985; Offner et al., 1986).

inlrm I 1

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I

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I E K N F V G I Y K L V D S K N F D O Y M K S t G V G F A I R ~ V A S M I K P l I l I E K N C O I l Y G K I H S l F K N ~ E l S N F ~ L G infton I1

inhm Ill

L -FW

I

I-FW

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FIG. I . Comparison of the primary structure of the three rat cytosolic FABPs. Standard single letter nomenclature for amino acids is used. The numbers refer to residue positions in L-FABP only. The arrows indicate the positions corresponding to the location of the three introns within the transcription unit of the L-FABPgene. The boxed residues comprise tandemly repeated segments within the primary structure of the individual FABP determined by RELATE program analysis (see text for details). Data are derived from Gordon et a/. (1985). Alpers et a / . (1984). Lowe et a / . (1985). Gordon and Lowe (1985), Sacchettini et a / . (1986). and Sweetser et a/. (1986).

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NATHAN M. BASS

Lowe et al. (1985) have performed careful analyses of the primary structure of L-FABP in order to identify domains of amphipathic a-helical structure similar to the type present in apolipoproteins A1 and AIV and in the carboxy-terminal region of serum albumin. These domains are thought to be of importance in bestowing the lipid binding properties of these proteins, and are characterized, when viewed end on with the amino acid side chains projecting radially, by oppositely situated hydrophobic and hydrophilic (acidic) surfaces. In the apolipoproteins, these domains occur as tandemly arrayed 11 or 22 amino acid long repeats. Using the program RELATE to identify internal repeats within L-FABP, Lowe et al. (1985) identified two tandemly arrayed sequences consisting of residues 23-36 and 37-49. A second pair of tandem repeats exhibiting significant homology with the first set spanning residues 23-49 was identified at residues 87-1 12 (Fig. 1). These tandem repeat segments were also identified in human L-FABP and suggest a phylogeny involving two ancestral gene duplication events. Further analysis, however, failed to reveal amphipathic a-helical structure within these repeated segments or within other domains of L-FABP. Gordon and Lowe (1985) have thus inferred that the structural basis for L-FABP fatty acid interactions must be different from that of albumin and the apolipoproteins. In contrast, however, Chan et al. (1989, using a comprehensive analytical strategy involving the calculation of helical hydrophobic moment, hydropathy, and a-helical probability for human L-FABP, have concluded that this protein is highly amphiphitic. This issue has considerable bearing on the question of not only how and where within its structure L-FABP binds fatty acids, but also whether or not it is disposed toward interacting with lipid-water interfaces, and whether or not it requires information about the tertiary structure of L-FABP for its resolution. Analysis of I-FABP and M-FABP using RELATE (Gordon and Lowe, 1985; Sacchetini et al., 1986) reveals tandem repeats in these proteins as well (see Fig. l), and in the case of I-FABP, sequences related to its NH,terminal domain as well. As shown in Fig. 1, however, the position and size of the related sequence blocks within members of the FABP family are not consistent. Nevertheless, considerable sequence homology exists between the three rat cytosolic FABPs (Fig. 1) as well as between the FABP and a group of similar sized proteins comprising mouse adipocyte p422 protein, myelin P2 protein, cellular retinoic acid binding protein (CRABP), and cellular retinol binding protein (CRBP) (Takahashi et al., 1982b;Bernlohretal., 1984;GordonandLowe, 1985;Sundelinetal., 1985). The comparative relatedness of these proteins derived from computer matching of their amino acid sequences is shown in Table 11. The greatest degree of structural similarity between the proteins compared in Table I1

149

T H E CELLULAR FATTY ACID BINDING PROTEINS TABLE I1 BETWEENTHE FABP AND OTHER PROTEINS‘

SEQUENCE COMPARISON SCORES

L-FABP Human L-FABP I-FABP M-FABP Mouse p422 protein Human myelin Pz protein Bovine CRABPd Rat C R B F Bovine SCP, Rat serum albumin

41.354 5.720 5.584 4.953 4.630 9.154 6.456 - 1.103 - 0.658

I-FABP M - F A B F -

-

8.761 11.860 11.265 10.359 7.440 1.643 - I .397

-

29.654 22.659 1 13 7 3 9.864

-

-0.136

“The RELATE algorithm and mutation data matrix (250 PAMS) were used to compare sequences between the proteins in the table. The SD values in the table were derived by comparing the RELATE comparison score for two real sequences with the mean score from multiple comparisons of random sequences having the same amino acid compositions as the real sequences. An SD score of 3 suggests a statistically significant relationship (probability of a chance similarity is less than 1 in 10’). An SD score of 10 indicates that the probability of sequence similarities being due to chance alone is less than I in Id’. A negative score indicates that the score obtained for comparing random shuffled sequences was greater than the score obtained for real sequences. bData from Gordon and Lowe (1985). ‘Data from Sacchettini el a/. (1986). dCellularretinoic acid binding protein. ‘Cellular retinol binding protein.

is th t found between orthologous rat and human L-FABP (SD core 41.354; 82% sequence homology). The ranking of structural relationships between M-FABP and the other proteins in Table I1 is of the order: p422 protein (SD score 29.654; 62% sequence homology) > mylein P, protein > CR4BP > CRBP > I-FABP > L-FABP (SD score 5.584; 36% sequence homology). It is also interesting to note that no significant homology exists between the FABP and serum albumin or SCP,. Sweetser et al. (1986) have recently added a further dimension to the understanding of the structural relationship between the FABP and homologous proteins. These workers have recently isolated and determined the nucleotide sequence of the L-FABP gene. The L-FABP transcription unit spans 3790 nucleotides and contains four exons (1 15, 173, 93, and 121 base pairs) interrupted by three introns (1454, 1224, and 610 base pairs). Of the four exons, only exon 1 (which encodes the 23 amino acid NH,-terminal portion of the protein) encodes a sequence with significant homology to all the other members of the extended “FABP family” (IFABP, M-FABP, CRBP, CRABP, myelin P, protein, and p422 protein).

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It is attractive to speculate that the conservation of this primary structural region between several members of a low-molecular-weight“lipid binding protein” family could point to its importance possibly in conferring a common structural domain essential for hydrophobic binding interactions, but no direct evidence for this exists as yet. Chan et al. (1985) have proposed an evolutionary tree for FABP-related proteins based upon analysis of mRNA and protein sequences. They estimate that an ancestral gene diverged approximately 1 billion ( lo9)years ago into L-FABP/I-FABP and p422/myelin P2 precursors. The initial divergence of L-FABP and I-FABP genes would have taken place about 650 million years ago, while the genes for p422 and P, diverged about 490 million years ago.

IV. Tissue Expression of the FABP Rabbit antibodies raised against the different rat cytosolic FABP show no immunological cross-reaction among these proteins (Said and Schultz, 1984; Bass and Manning, 1986), and have thus permitted their specific immunoassay in the cytosol of rat tissues (Ockner and Manning, 1974; Ockner et al., 1982; Bass et al., 1985a; Bass and Manning, 1986). The tissues containing the most abundant quantities of the FABP are found in liver (L-FABP, 3% of cytosol protein), intestine (L-FABP and I-FABP, both 1-2% of cytosol protein), and heart muscle (M-FABP, 5% of cytosol protein). The relative abundances of the three cytosolic FABP in a wide range of rat tissues are shown in Table 111. I-FABP expression is the most restricted, being present only in intestinal epithelial cells, while M-FABP is expressed in a broad range of tissues. Haq et al. (1982) reported the purification of L-FABP from adipose tissue. Work from other laboratories (Bass and Manning, 1986; Brecher et al., 1986), however, has indicated that both L-FABP and M-FABP are expressed in minute quantities in adipose tissue. It is interesting in this regard to note that mouse adipocyte p422 protein, although structurally closely related to rat M-FABP, is expressed exclusively in mouse adipocytes (Bernlohr et al., 1985), raising the question of whether or not a unique adipocyte FABP exists in the rat as well. In the small intestine, both L-FABP and I-FABP are most abundant in the jejunum and relatively less abundant in ileum (Ockner and Manning, 1974; Bass et al., 1985a). On immunohistochemical staining using specific antibodies, both proteins are present predominantly in the enterocytes at the tips of the intestinal villi and virtually absent from crypt cells (Shields et al., 1986). This pattern of distribution of the FABP in the intestine is

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TABLE 111 TISSUE EXPRESSION OF THE FABP“ Tissue

M-FABP

Relative % L-FABP

I-FABP

~

Heart Liver Small intestine Jejunum Ileum Large intestine Stomach Pancreas Kidney Testis Ovary Adrenal Striated muscle Soleusb Red vastus lateralisb White vastus lateralisb Abdominal muscles Mammary gland Brain Lung Adipose tissue Spleen Plasma

100

0

0

100

0 0 0 7.2 0 4.2 7.3 5.7

0 75 56 15 5.8 45

2.0 1.1

I .o 0 0

0 0

86 33 17 1.4 I .O 0 0 0 0

100

0 0 0 0 1.o 0 0 1.O 0 0

0 0 0 0 0 0 0 0 0 0

67 24 4.9 0 0 0 0 0

T h e FABPs were determined in 105,000 g supernatants of tissues by immunoassays employing antibodies specific to each of the three proteins. Tissues with the highest cytosol concentrations (pg FABP/mg cytosol protein) for each of the FABPs (liver. jejunum, and heart muscle for L-FABP. I-FABP, and M-FABP, respectively) were assigned a value of 100% and values for other tissues were expressed as relative percentages. Data are from Bass and Manning (1986) unless otherwise specified. bMiller er al. (1986). ‘Jones er a/. (1986).

identical with the normal anatomical localization of the processes of absorption and esterification of dietary fat in the intestine (Johnston, 1969; Hoving and Valkema, 1969). L-FABP is also not uniformly expressed in all cells throughout the hepatic lobule, and has been localized by immunohistochemical staining to the hepatocytes in acinar zone 1, i.e., hepatocytes in close proximity to the portal venules (Fig. 4; Bass et al., 1985~). The significance of this lobular “gradient” of L-FABP in the liver is uncertain, and is discussed further in Section VII,C.

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The distinctive pattern of tissue expression of the cytosolic FABPs lends support to the general notion that they may perform structure-dependent specialized functions in fatty acid transport and/or metabolism. L-FABP and I-FABP are both expressed in tissues in which both oxidation as well as extensive esterification of fatty acids to triglyceride occur. The significance of the dual expression of both proteins in the intestine is unknown, although a role in compartmentalization of fatty acids has been suggested (Alpers et al., 1984; Bass, 1985). The tissue distribution of M-FABP suggests a role for this protein as an intracellular carrier of fatty acids destined predominantly for oxidative catabolism. This is most strongly supported by the finding that M-FABP in skeletal muscle is most abundant in soleus and red vastus lateralis, muscles that contain the highest red fiber content (Miller et al., 1986; Brecher et al., 1986). On the other hand, the abundance of M-FABP in mammary gland (Table III), in which triacylglycerol biosynthesis is the predominant process utilizing fatty acids, seems contrary to this hypothesis (Jones et al., 1986). Dempsey et al. (1985) have reported finding substantial quantities of SCPIL-FABP in the serum as well as in several tissues including heart muscle, kidney, and lung. Since L-FABP mRNA is significantly expressed only in small intestine and liver (Gordon et al., 1983, it was postulated that SCP/L-FABPis secreted by the liver and intestine into the circulation from where it is removed by several tissues lacking a suitable coding mRNA for SCP/L-FABP and incorporated into their pool of cellular proteins. This proposal contains several concepts that are biologically novel. As discussed in Section 111, the posttranslational processing of L-FABP is not that of a secretory protein but typical for a cytoplasmic protein. In addition, others have consistently failed to find L-FABP in the serum of normal rats (Vincent et al., 1985; Bass and Manning, 1986) or in tissues other than liver and intestine (Bass and Manning, 1986). The findings reported by Dempsey ef al. (1985) are particularly puzzling in that their antibodies to SCP/L-FABP cross-react completely with L-FABP (M. E. Dempsey, personal communication; J. I. Gordon, personal communication). It would therefore seem that the antibodies to SCP/L-FABP used by Dempsey et al. (1985) in their immunoassays recognize other proteins apart from L-FABP, and the precise nature of the antigen that these workers measure as SCP/L-FABP in serum and heart muscle needs to be determined. Immunocytochemical studies of the subcellular distribution of L-FABP and I-FABP by both light and electron microscopy have localized both proteins within the cytoplasm of hepatocytes and enterocytes (Capron et al., 1979; Bass et al., 1985c; Shields et al., 1986). Although neither protein

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was seen within cellular membranes or organelles, L-FABP and I-FABP in the intestine of suckling rats were localized most intensely around the Golgi apparatus area (Shields et al., 1986), while L-FABP in hepatocytes was noted to be most densely accumulated in the cytosol surrounding the smooth endoplasmic reticulum and mitochondria (Capron et al., 1979). A similar distribution of cytosolic glutathione S-transferase B was found in the studies of Capron et al. (1979), and it is thus unclear whether the subcellular geographic distribution observed for L-FABP represents an in vivo gradient of functional significance o r an artifact induced during fixation and processing of tissue sections. Fournier and Rahim (1985) have localized M-FABP by means of the immunogold method to cardiac myocyte myofibrils, mitochondria, interorganellar cytosol, and intercellular spaces, as well as capillary endothelial cells and red blood cells. From these findings, the authors have extrapolated the concentration of M-FABP within subcellular compartments and have concluded that the distribution of M-FABP displays marked gradients from the myofibrillar to the mitochondria1 and interorganellar compartments. However, the caveat regarding fixation artifacts discussed above must also be invoked regarding the morphological data presented by Fournier and Rahim (1985), and the possibility that M-FABP does indeed form gradients between subcellular structures and exists within mitochondria requires further evaluation. lmmunocytochemical studies provide no evidence for the presence of cytosolic FABP or cross-reacting proteins incorporated within cellular membranes. However, several lines of evidence support the notion that the FABP may physically interact with cellular membranes in a reversible fashion. Ockner and Manning (1974) found 15% of total intestinal I-FABP associated with washed particulate cell material including nuclei, plasma membranes, mitochondria, and microsomes. Gordon et al. (1983) found that the inclusion of microsomes during L-FABP translation by an ascites cell-free translation system protected the protein from trypsin cleavage, while Burton and Bloch (1985) have demonstrated saturable binding of SCP/L-FABP to microsomal membranes that appeared to depend upon the integrity of microsomal proteins but not phospholipids. LPM-FABP appears to be an integral membrane protein and has bden localized by immunofluorescence to the plasma membranes enclosing all surfaces of hepatocytes, the brush border and lateral border membranes of enterocytes in both villi and crypts, the intercalated disks of heart muscle, and to tissue macrophages (Stremmel et al., 1985a,b). It is unknown whether LPM-FABP is also present in adipocytes. Abumrad et al. (1984), however, have isolated an 85-kDa protein from adipocyte membranes that

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may be involved in fatty acid transport. Since bifunctional stilbene reagents were used to isolate this protein, it is conceivable that it represents a dimer of the 40-kDa LPM-FABP (Stremmel et al., 1985b).

V. Ligand Binding to FABP

A. ENDOGENOUS LIGANDS About 60% of cytosolic long chain fatty acids are associated with LFABP, and these show a substantial enrichment in long chain unsaturated fatty acids including the essential fatty acids linoleic and arachidonic acids over the proportions found in serum (Rustow et al., 1978; Burnett et al., 1979; Ockner et al., 1982). This has suggested that L-FABP may act as an acceptor for fatty acids released from phospholipids by phospholipase A2, and that it may furthermore perform an important storage function through maintaining an intracellular pool of essential fatty acids for the synthesis of phospholipids, prostaglandins, and leukotrienes. L-FABP isolated from undelipidated liver cytosol can be separated by ion exchange chromatography and isoelectric focusing into three to five isoforms (Ketterer et al., 1976; Trulzsch and Arias, 1981b; Ockner et al., 1982; Takahashi et al., 1983) that differ both qualitatively and quantitatively in bound endogenous long chain fatty acids (Takahashi et al., 1983; Bass, 1985). The near-neutral isoform of L-FABP (pZ 6.9) contains the least amount of bound endogenous fatty acids, whereas the more acidic isoforms (pZ 6.0, 5.6, and 5.2) contain fatty acids in quantities of approximately 1 moVmol protein (Bass et al., 1984b; Bass, 1985). Delipidation of L-FABP leads to a virtual abolition of the relatively acidic isoforms of the protein and an increase in the pZ6.9 species (Haunerland et al., 1984; Bass et al., 1984b). Thus, the charge heterogeneity of L-FABP appears to be largely a consequence of bound endogenous fatty acids that may alter the surface charge of the protein by both contributing carboxyl groups and inducing conformational changes in the protein in a manner similar to the effects of fatty acids on serum albumin (Basu et al., 1978). Rustow et al. (1982) have found phospholipids, monoglycerides, diglyceride, and triglyceride in large quantities, as well as small amounts of cholesterol endogenously associated with L-FABP, but others have failed to confirm this (Burnett et al., 1979; Takahashi et al., 1983; Bass, 1985). Nonlipid ligands that have been bound endogenously associated with LFABP include bilirubin in jaundiced Gunn rats (Levi et al., 1969) and heme (Billheimer and Gaylor, 1980). M-FABP contains much smaller amounts of endogenous fatty acids than L-FABP (Bass and Manning, un-

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published data), while M-FABP from bovine heart shows no change in isoelectric point as a function of bound fatty acids (Jagschies et al., 1985). B. STUDIES OF EXOGENOUS LIGAND BINDING A variety of methods have been used to characterize the specificity, affinity, and stoichiometry of ligand binding to the FABP. These have included gel filtration assays (Ockner et al., 1972a; Mishkin et al., 1972; Levi et al., 1969; Mishkin and Turcotte, 1974a; Lichter et al., 1976),equilibrium dialysis (Ketterer et al., 1976), circular dichroism (Kamisaka et al., 1975; Tipping et al., 1976; Keuper et al., 1985), difference spectrophotometry (Tipping et al., 1976; Vincent and Muller-Eberhard, 1985), electrophoretic titration (Haunerland et al., 1984), fluorescent dye competitive displacement (Takikawa and Kaplowitz, 1986), and quenching of intrinsic protein fluorescence (Vincent and Muller-Eberhard, 1985), as well as assays involving partition of ligands between pure FABP and charcoal (Warner and Neims, 1975), Lipidex-1000 (Glatz et al., 1984, 1985a,b; Bass, 1985), and multilamellar liposomes (Brecher et al., 1984; Offner et al., 1986). It is well established that only fatty acids with chain lengths of 316 carbons bind to L-FABP (Ockner et al., 1972a; Mishkin et al., 1972; Rustow et a / . , 1978; Bass, 1985) with apparent affinities (&) of the order of 0.5-1 pA4 (Bass, 1985; Glatz et al., 1985a,b; Vincent and Muller-Eberhard, 1985). Values for Kd of the order of 5-12 FM have also been reported for palmitate and oleate (Ketterer et al., 1976; Brecher et al., 1984) and may reflect differences in methodology or failure to delipidate the protein prior to performing binding assays (see Glatz and Veerkamp, 1985). In general, unsaturated long chain fatty acids have been found to bind more avidly to L-FABP than saturated long chain fatty acids on gel filtration assays (Ockner et al., 1972a; Mishkin et al., 1972). However, equilibrium binding assays reveal only subtle differences in Kd as a function of the degree of fatty acid saturation (Bass, 1985; Glatz et al., 1985a,b). Both long chain acyl-carnitine and acyl-CoA esters bind to L-FABP at the fatty acid binding site, but it is still unclear whether acyl-CoA esters bind with greater (Mishkin and Turcotte, 1974b; Ketterer el al., 1976) or lesser (Bass, 1985) affinity than their corresponding unesterified free fatty acids. Although a function for L-FABP as a sterol carrier protein with a role in cholesterol metabolism has been reported (Dempsey, 1975, 1984; Dempsey et al., 1985), attempts at demonstrating binding of cholesterol to L-FABP have been uniformly unsuccessful (Ockner et al., 1972a; Mishkin et al., 1972; Haunerland et al., 1984; Bass, 1985). Schroeder et al. (1985) have, however, reported that the fluorescent sterol, cholesta-

156

NATHAN M. BASS

trienol, bound to SCP/L-FABP with a K , of 0.57 p M in a 1: 1 molar ratio to the protein. Furthermore, cholesterol was shown to decrease cholestatrienol binding to SCP/L-FABP, but it is uncertain whether this represented the result of competitive displacement or micellar extraction. Burton and Bloch (1985) were also unable to detect lathosterol binding to SCP/L-FABP by equilibrium dialysis. However, several other molecules with sterol structures have been reported to bind to L-FABP. These include steroid hormones and their metabolites and bile acids (Ketterer et al., 1976; Takikawa and Kaplowitz, 1986), although in general their affinities were lower than those observed for long chain fatty acids. As regards other classes of lipids, binding of lysophosphatidlylcholine to L-FABP, but not M-FABP, has been reported (Burrier and Brecher, 1986) and there is some evidence to suggest that L-FABP may form complexes with phosphatidylserine (Burton and Bloch, 1985) but not with phosphatidylcholine (Burton and Bloch, 1985; Keuper et al., 1985) or cholesteryl esters (Keuper et al., 1985). Nonlipid ligands reported to bind to L-FABP include bilirubin, bromosulfophthalein (Levi et al., 1969; Ockner et al., 1972a; Mishkin et al., 1972; Kamisaka et al., 1975; Ketterer et al., 1976; Warner and Neims, 1975; Vincent and Muller-Eberhard, 1985; Bass, 1985), carcinogens (Ketterer et af., 1976), thyroid hormones (Warner and Neims, 1975; Lichter et al., 1976), retinoids (Vincent and Muller-Eberhard, 1985), the fluorescent dyes ANS and TNS (Takikawa and Kaplowitz, 1986; Keuper et al., 1985), choline (Trulzsch and Arias, 1981a), hexachlorophene (Warner and Neims, 197% and radiographic contrast dyes (Sokoloff et al., 1973). Reported Kd for these ligands are, for the most part, at least an order of magnitude greater than the Kd values documented for fatty acids. In the case of bilirubin, reported & values range from 0. l to 50 p M (Ketterer et a / . , 1976; Warner and Neims, 1975), but a lesser affinity for this ligand compared to oleate for binding to L-FABP is apparent from competitive inhibition studies (Bass, 1985). The only non-fatty-acid ligand that has been consistently found to bind to L-FABP with affinities higher than fatty acids is heme, with Kd ranging between 0.12 and 1.5 p M (Tipping et al., 1976; Billheimer and Gaylor, 1980; Bass, 1985; Vincent and Muller-Eberhard, 1985). Several other porphyrins also bind to L-FABP with K, values ranging from 0.43 p M for protoporphyrin to 9.56 p M for coproporphyrin (Vincent and Muller-Eberhard, 1985). Binding of fatty acids to I-FABP and M-FABP has been studied only to a limited extent, but available data suggest that the affinities of these proteins for long chain fatty acids are of the same order found for LFABP, although the molar ratio of fatty acids bound per mol protein has been consistently less than comparative ratios for fatty acid binding to LFABP (Glatz et al., 1985; Offner et al., 1986; Bass, unpublished data). It

THE CELLULAR FATTY ACID BINDING PROTEINS

157

is interesting to note in this regard that Fournier and Rahim (1983, 1985) and Fournier et a / . (1983) have reported that M-FABP undergoes concentration-dependent self-aggregation, which competes with fatty acid binding and which may account for the observed lower capacity of MFABP to bind fatty acids. The stoichiometry of fatty acid binding to the FABP is an unsettled issue at present. Several studies have reported ligand associations with L-FABP of 1 mol/mol protein at saturating concentrations of ligand (Ketterer et a / . , 1976; Lunzer et a / . , 1977; Glatz et a / . , 1984; Brecher et a / . , 1984; Vincent and Muller-Eberhard, 1985; Glatz et al., 1985a,b; Bass, 1985). Recently, however, Offner et a / . (1986), using a binding assay employing liposomes as fatty acid donors, have found saturation of oleate and palmitate binding to both L-FABP and M-FABP at 2:1 molar ratios of fatty acid to protein. These authors point out that FABP concentration is overestimated by conventional protein assays and that this could lead to underestimation of the number of binding sites per molecule of FABP. However, others have also noted the discrepancy between FABP concentration determined by colorimetric assays and aminoacyl mass determinations (Ockner et al., 1982; Glatz et al., 1985a,b), and even with compensation for this phenomenon, have derived 1: I stoichiometries for saturable binding of fatty acids to L-FABP (Bass et al., 1984b; Bass, 1985; Glatz et a / ., 1985a,b). Some reconciliation of stoichiometric differences may be afforded by findings from the author’s laboratory, in which, as shown in Fig. 2, binding of long chain fatty acids to L-FABP reveals both 1: I saturable as well as nonsaturable components of binding (Bass et al., 1984b; Bass, 1985). The assay of Offner et al. (1986) yields lower affinities for fatty acid binding to the FABP than those found by Bass (1985) and Glatz et al. (1989, and may thus not differentiate between high- and lowaffinity binding interactions. In elegant studies employing electrophoretic titrations of L-FABP complexed with radiolabeled fatty acids and the fluorescent fatty acid derivative 16-anthroyloxy palmitate (A16:0), Haunerland et al. (1984) found that 2 rnol of long chain fatty acid is bound per rnol of protein but that only 1 rnol of A16:O is accommodated per mol of protein. They concluded that L-FABP contains a single binding site capable of accommodating two long chain fatty acids; one bound mainly by ionic forces, the second mainly by hydrophobic interaction. When A16:O is bound, interaction of the second fatty acid may be prevented by the bulky anthracene group of A16:O completely blocking the hydrophobic binding region. In support of this, Keuper et al. (1985) have observed binding in close proximity of 2 rnol trans-parinaric acid/mol LFABP on circular dichroism spectroscopy. The requirement of both hydrophobic and ionic interactions for the binding of fatty acids to L-FABP is also apparent from the fatty acyl chain

158

NATHAN M. BASS

TOTAL OLEATE &M)

FIG.2. Binding of [‘4C]oleateto L-FABP. The binding assay contained 420 nm L-FABP and [‘4C]oleate in the concentrations indicated. Unbound fatty acids were removed using Lipidex-1000 (Bass, 1985). The binding data were analyzed by least squares nonlinear regression analysis, and were fitted best by an equation containing parameters for both saturable and nonsaturable components of binding. The solid line indicates total binding of oleate to L-FABP; the broken line indicates the nonsaturable component of binding.

length requirements for binding as well as from studies of the effect of pH on binding and the poor interaction of fatty acid methyl or ethyl esters with this protein (Ockner et al., 1972a; Brecher et al., 1984; Haunerland et al., 1984). Studies of fluorescent quenching (Keuper et al., 1985) and excited state lifetimes (Storch et al., 1986) of a series of long chain fatty acid anthroyloxy derivatives bound to L-FABP have further indicated that the mid-portion of the acyl chain is most deeply recessed in a hydrophobic “pocket,” whereas the head group and CH,-terminal lie closer to the surface of the protein. Since the NH,-terminal domains of the FABP as well as other homologous proteins show the greatest degree of sequence conservation (see Section 111), it has been speculated that this region of the molecule may be important in conferring lipid binding properties common to all the members of this protein family (Gordon and Lowe, 1985). However, a frameshift mutation induced in L-FABP, which altered the amino acid sequence from positions 104 to 117 and deleted the COOH-terminal 10 amino acids, led to loss of fatty acid binding properties (Lowe et al., 1984). Loss of binding in these studies is difficult to interpret as it could indicate either the importance of the COOH-terminal in formation of the binding site or that its deletion alters the general conformation of the protein and

THE CELLULAR FATTY ACID BINDING PROTEINS

159

thus perturbs a binding site formed at the NH,-terminus. Nevertheless, the technique of site-directed mutagenesis holds considerable promise for elucidating the molecular basis for FABPhigand interactions. A far greater specificity of ligand binding has been reported for LPMFABP compared to the cytosolic FABP. Stremmel et al. (1985b) have demonstrated binding of several long chain fatty acids to this protein, but no binding of bilirubin, bromosulfophthalein, taurocholate, phosphotidylcholine, or cholesteryl esters. VI. Regulation of the FABP

A. TURNOVER AND DIURNAL REGULATION The directly measured half-life of L-FABP degradation in liver is 3.1 days, which is close to the average value obtained for whole cytosol protein (Bass, et al., 1985b; Bass, 1985). This relatively slow turnover of L-FABP argues against any potential for this protein to undergo rapid modulation of its cellular content. However, Dempsey’s group (Dempsey, 1984; Dempsey et al., 1985) has reported that SCP/L-FABP undergoes a striking diurnal variation in hepatic content, ranging from 1.4 to 14.6% total liver protein from the beginning of a 12-hour light period to the mid-point of a 12-hour dark period. This diurnal change in SCP/L-FABP was found to be attributable to changes in protein synthesis alone, without changes in SCP/L-FABP mRNA abundance. These findings imply a half-life of less than 1 hour for the protein and regulation of its diurnal cycle at the level of translation. The diurnal cycle of SCP/L-FABP corresponds to the nocturnal feeding cycle of rats, and evidence was recently presented that it is mediated mainly by insulin (Hargis et al., 1986). The reported diurnal variation of SCP/L-FABP has also been taken as possible evidence for a role for this protein in regulating the biosynthesis of cholesterol which also undergoes a marked diurnal cycle (Dempsey , 1984; Hassan, 1986). Glatz et al. (1984), using a binding assay, initially also reported a two-fold diurnal variation in the tissue content of both LFABP and M-FABP, but this group has subsequently been unable to reproduce these findings, and has shown that the quantitation of cytosolic FABP content by fatty acid binding assay yields highly variable results depending upon the type of radiolabeled fatty acid used and the adequacy of albumin removal (Paulussen et al., 1986). Other workers, using either immunoassay (Bass et al., 1985b)or a binding assay for L-FABP (Wilkinson and Wilton, 1986, 1987), have found no diurnal variation in this protein. Because the difference between the finding of Dempsey’s group and others regarding L-FABP diurnal regulation is

160

NATHAN M. BASS TABLE 1V EFFECTOF DIURNAL LIGHT-DARK CYCLEON L-FABP“ Hornogenate

Cytosol Time

24.00 hours (rnid-dark)

06.00 hours 12.00 hours (mid-light)

(p,g/rng protein)

(&ng

protein)

(rng/g liver)

45.7 43.4

-L

3.1 4.0

8.9 8.8

2

2

2

1.1 1.3

5.3 5.7

46.0

f

0.4

8.4

2

0.1

5.0 2 0.1

2

1.1

* 1.4

(rng/g liver)

0.83 0.73

f

2

0.10 0.13

0.75 f 0.10

‘’Values represent means r SD for three female rats. Homogenates were prepared in 0. I M phosphate buffer. pH 7.6.0.15 M KCI. Cytosol represents the 105.000 g supernatant. Both cytosol and homogenate were diluted in phosphate-buffered KCI containing 0.1%Triton X-100prior to measurement of L-FABP by radial immunodiffusion assay.

so considerable, the author has recently performed additional experiments using female rats (employed in Dempsey’s studies) acclimated to a 12hour Iightll2-hour dark cycle for 2 weeks. The results of these experiments are shown in Table IV. No diurnal variation in the concentration or total hepatic content of L-FABP was found. A small decline in L-FABP expressed as mg/g liver was seen between the mid-dark and light periods attributable entirely to diurnal changes in liver weight, a previously welldocumented phenomenon (Seifert, 1980). No acceptable explanation is yet at hand for the differences in fundamental regulatory behavior of what appears to be an identical protein in the hands of Dempsey’s group and other workers, although, as discussed earlier, it seems likely that the SCP/L-FABP immunoassay is not specific for L-FABP. It is interesting that SCP, also lacks diurnal variation (Kharroubi et al., 19861, and thus cannot be implicated as a possible crossreactant in the SCP/L-FABP immunoassay, which might account for Dempsey’s findings. B. DIETARY REGULATION Following a 48-hour fast, the total content of L-FABP in liver declines 3670% in parallel with total liver cytosol protein (Stein et al., 1976; Brandes and Arad, 1983; Bass et al., 1985b). Thus, L-FABP concentration with respect to cytosol protein is minimally affected by acute starvation (Bass et al., 1985b). Refeeding a sucrose-rich diet for 24 hours after acute starvation leads to little change in either the total liver content or cytosolic concentration of L-FABP (Brandes and Arad, 1983; Bass et al., 1985b). Thus, in keeping with its relatively slow rate of turnover in liver, L-FABP concentration does not undergo significant modulation with transitions from the fed to fasted to refed states during which rapid, major transitions

THE CELLULAR FATTY ACID BINDING PROTEINS

161

in hepatic fatty acid flux, fatty acid oxidation, and triglyceride biosynthesis occur (Brindley, 1978). On the other hand, it is evident from these findings that the cytosolic concentration of L-FABP is well preserved under the conditions of increased protein catabolism (Dice et al., 1978) and fatty acid flux that occur during starvation. Similar "sparing" of I-FABP in the intestine has been observed following a 3-day fast (Ockner and Manning, 1974). A lack of hepatic L-FABP responsiveness to acute transitions between different modes of fatty acid utilization by the liver is also apparent from work by Gordon et al. (1985) which examined the changes in liver and intestinal L-FABP and I-FABP mRNA from fetal to adult stages of life in the rat (Fig. 3). As shown in Fig. 3, L-FABP mRNA in liver increases A

Fetal

I Neonatal

-

B

Liver

FABP cDNA Smoll Intestine

Intestinal FABP c D N A

-

5 17 19 21 1

4

8

FTG"l:'ver

DAYS OF DEVELOPMENT

FIG. 3. Developmental regulation of (A) L-FABP and (B) I-FABP mRNA in rat small intestine and liver. The data shown were obtained from quantitative densitometric tracings of dot blots containing total cellular RNA isolated from fetal, neonatal, and adult rat small intestine and liver that had been probed with either '*P-cloned L-FABP (A) or I-FABP (B) cDNA. Reproduced from Gordon e? a / . (1985) with permission from the Journal ofBiologicul Chemistry and the authors.

162

NATHAN M. BASS

abruptly at birth concomitant with the commencement of the suckling period. However, no change in hepatic L-FABP mRNA is observed during the transition from the suckling to the weanling state which occurs at about day 14, a period which is characterized by a marked reduction in hepatic fatty acid oxidation (Henning, 1981). Similar changes in L-FABP protein concentration in the liver during early development have been reported (Sheridan et al., 1987). The expression of L-FABP mRNA in intestine also increases abruptly at birth and continues to rise until the end of the suckling period (day 14), whereafter levels decline during weaning up to 35 days followed by a secondary rise during maturation. Although similar developmental changes are observed for I-FABP mRNA, these are far more subtle during the suckling and weanling periods than the changes in intestinal L-FABP mRNA during this period. These studies suggest that the genes for intestinal L-FABP and I-FABP are both induced by dietary fat content, although there are quantitative differences in their responsiveness. Studies of the effects of feeding diets rich in either fat or carbohydrate on cytosolic FABP measured by immunoassay in adult rats also bear out this conclusion (Table V). Several aspects of the dietary regulation of the FABP are apparent from the data in Table V: (1) Diets rich in fat lead to modest increases in MFABP in heart, and L-FABP in liver and small intestine. The increase for L-FABP is most marked in the ileum. In the case of I-FABP, an increase in response to fat is seen only in the ileum; (2) a sucrose-rich diet has no TABLE V EFFECTOF DIETARY FATA N D CARBOHYDRATE CONTENT ON FABP CONCENTRATION"

FABP L-FABP

I-FABP

Tissue Liver

36

Jejunum

23

Ileum

10

Jejunum

15

Ileum M-FABP

Mean control values (ugh cytosol protein)

Heart muscle

7

4.3 (mglg tissue)

Diet High fat High CHO High fat High CHO High fat High CHO High fat High CHO High fat High CHO High fat

Relative change ( x control)

Reference

1.4 I .o I .3 0.7 I .5 I .o I .o

Bass and Manning (unpublished data)

Ockner and Manning ( 1974)

I .4 1 .o I .3

Fournier and Rahim (1985)

0.64

"All determinations were made by immunoassay. Values are based on means for 3-6 rats. Control diets contained approximately 5% fat (w/w). High-fat and high-carbohydrate (CHO) diets contained 20-38% vegetable oil (wlw) or 60% mcrose (w/w). Animals were maintained on the diets 21-31 days.

THE CELLULAR FATTY ACID BINDING PROTEINS

I63

effect upon L-FABP in liver and ileum or I-FABP in ileum, but leads to a fall in both L-FABP and I-FABP in jejunum. It would therefore seem reasonable to conclude that the levels of L-FABP in liver and both LFABP and I-FABP in the ileum of mature animals are maintained at a constitutive minimum under conditions of normal fat intake, and are inducible by marked increases in dietary fat intake. L-FABP in jejunum is also induced by fat feeding, but levels of this protein as well as of jejunal I-FABP fall with a low-fat (high-sucrose) diet, implying that both proteins are moderately induced in the jejunum under conditions of normal fat intake. Indeed, under normal dietary conditions, I-FABP in the jejunum appears to be expressed at o r near maximum levels. The lack of hepatic L-FABP response to a sucrose-rich diet (Table V) further suggests that the substantial increase in hepatic lipogenesis that accompanies this diet is incapable of increasing the expression of this protein above its constitutive minimum level. Thus, although hepatic LFABP levels appear to be responsive to the flux of exogenous fatty acids, this protein does not appear to be induced by an increased flux of endogenous fatty acyl-CoA derived from lipogenesis. Others, using binding assays to measure liver L-FABP, have noted increases in this protein on feeding diets rich in fat and carbohydrate (Haq and Shrago, 1983) or decreases on feeding a linoleate-rich diet (Herzberg and Rogerson, 1981). However, binding assays are less reliable than immunoassays for FABP quantitation and are influenced substantially by endogenous ligands (Glatz and Veerkamp, 1983, as well as the choice of radiolabeled ligand (Paulussen et al., 1986). The mechanism by which FABP levels respond t o changes in dietary fat intake is unknown. Possibilities include direct nutrient interactions with the genome o r the proteins (Castro and Towle, 1986) o r hormonally mediated effects upon the synthesis and/or degradation of the FABP.

C. HORMONAL REGULATION Although a lipogenic diet does not result in the induction of hepatic LFABP, a limited body of data suggests that insulin (Brandes and Arad, 1983) and thyroid hormones (Reyes et al., 1971), both of which are known to induce enzymes responsible for lipogenesis (Sugden et al., 1985; Towle and Mariash, 1986), are also required for the expression of L-FABP in liver at constitutive levels. The cytosolic concentration of L-FABP is 1.6-1.7 times higher in the livers of mature female rats than in males (Ockner ef al., 1979, 1982; Bass et al., 1985a), whereas no sex difference in either L-FABP o r I-FABP in intestinal tissue is apparent (Bass et al., 1985a). The sex difference in hepatic L-FABP may be of importance in determining sex differences in fatty acid utilization (discussed in Section VII,C,2) and is completely

164

NATHAN M. BASS

reversed by a combination of gonadectomy and administration of estrogen to male and testosterone to female rats at 30 days of age (Ockner et al., 1980). Gonadectomy alone of immature rats does not of itself affect the sex difference in L-FABP to an appreciable extent, suggesting the possibilities that extragonadal sex steroids may also govern the sex difference seen in mature animals. The higher concentration of L-FABP in female livers results from increased synthesis without alteration in the fractional rate of degradation of the protein and is accompanied by an increase in L-FABP mRNA (Bass et al., 1985a). It is interesting that although intestinal L-FABP shows no sex difference in concentration, the mRNA specifying this protein is 1.5fold higher in female than in male intestine. However, L-FABP in female intestine also appears to undergo more rapid degradation (Bass et al., 1985a); thus, the protein does not accumulate in female intestine above the concentrations found in males. It is unclear at present whether or not the increased mRNA abundance and resultant increased synthesis of L-FABP in females are the result of increased gene transcription and/or mRNA stabilization. Furthermore, it is also unknown whether sex steroid hormones per se are the primary inducers of L-FABP, or whether their effects are mediated by other hormones (e.g., Mandour et al., 1977) or by sex differences in the hepatic flux of free fatty acid determined by other sex-related differences in hepatic fatty acid metabolism. L-FABP in female livers also shows a more uniform distribution in hepatocytes throughout the hepatic lobule which contrasts with the predominantly periportal distribution of the protein in male livers (Bass et al., 198%). In this regard, the induction of L-FABP in females shows some analogy to the induction of the FABPs in intestine by fat feeding, i.e., recruitment of expression occurs in cells that normally show minimum constitutive expression of the protein. It is unfortunate that this observation cannot resolve with certainty the mechanism of L-FABP modulation by sex-related factors because recruitment may be a general process in L-FABP modulation by a variety of factors and not necessarily indicative of a final common pathway of regulation by fatty acids. D. PEROXISOME PROLIFERATORS Administration of several plasma lipid-lowering agents including clofibrate, nafenopin, tiadenol, and phthalic acid esters has been shown to increase L-FABP concentration in liver (Fleischner et al., 1975; Renaud et al., 1978; Kawashima et al., 1982; Bass et al., 1985a; McTigue et al., 1985) and in intestine (Bass et al., 1985a; Kawashima et al., 1985). All these agents share in common the ability to produce a striking proliferation in the tissue content of peroxisomes, and lipid-lowering drugs that do not

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165

induce peroxisomes, e.g., probucol, also fail to induce L-FABP (Kawashima et al., 1982). Moreover, a strong correlation exists between the induction of peroxisomal p-oxidation by peroxisome proliferators and the magnitude of L-FABP increase (Kawashima ef al., 1983). Clofibrate treatment leads to increases in L-FABP concentration in liver, jejunum, and ileum to values 2-, 2.6-, and 3-fold higher, respectively, than control levels (Bass and Ockner, 1987). Maximum induced levels of the protein are reached by 10 days in liver and 3 days in the intestine (Bass, 1985). The induction of L-FABP by clofibrate occurs as a result of increased synthesis of the protein secondary to increases in the abundance of its mRNA without alteration in its rate of degradation (Bass et al., 1985a). I-FABP mRNA and protein abundance are also increased in the intestine with clofibrate treatment, yet only by a comparatively modest 25% above control values (Bass et al., 1985a). Although a striking relationship between peroxisome proliferation and L-FABP has been observed, the constitutive expression of L-FABP appears to be independent of the assembly of functional peroxisomes, as normal levels of L-FABP are found in the livers of infants with the Zellweger syndrome (Bass and Moser, unpublished data), in spite of the absence of peroxisomes in this disorder (Moser et al., 1984). Clofibrate does not bind to L-FABP (Vincent and Muller-Eberhard, 1985; Bass and Ockner, 1987)and its effects on the liver may be mediated by a specific receptor (Lalwani et al., 1983). Furthermore, it is unclear whether L-FABP is induced by peroxisome-proliferating agents as a result of their direct interaction with the genome in which L-FABP may exist as part of a “peroxisomal gene domain” (Watanabe et al., 19851, or whether this protein is induced secondary to an increase in free fatty acid flux generated by increased peroxisomal p-oxidation. Clofibrate administration also results in recruitment of L-FABP expression in hepatic centrizonal cells, a phenomenon similar to that observed in female rats (Bass ct al., 1985~).Because clofibrate leads to induction of L-FABP in the intestine, it is clear that a mechanism involving fatty acid flux would not depend upon dietary fatty acids since food intake between treated and control animals is identical (Bass et al., 1985a). Such a mechanism would therefore have to depend upon a marked increase in fatty acid delivery to the intestine from the plasma. This, however, seems an unlikely explanation. The magnitude of L-FABP induction by peroxisome proliferators is also consistently greater than that seen with prolonged high-fat feeding (Table V). Furthermore, the responses of L-FABP and I-FABP to fat feeding, although different, are not quite as divergent as the responses of these two proteins to clofibrate treatment. Thus, currently available data strongly suggest that the mechanism of clofibrate induction of the FABP is unlikely to be mediated by fatty acid flux.

166

NATHAN M. BASS

E. OTHERINFLUENCES L-FABP levels in liver are increased 1.5- to 2-fold by feeding cholestyramine (Kempen et al., 1983) and alcohol (Pignon et al., 1986), both of which also result in increased hepatic triacylglycerol synthesis, and in the case of cholestyramine, cholesterol biosynthesis as well. Marked reduction in hepatic L-FABP has been observed in mice with experimentally induced porphyria (Vincent et al., 1986), but it is unknown to what extent this was related to hepatocellular damage. VII. The Function of the Cellular FABP A. THEEFFECT OF FABP ON ENZYME ACTIVITY IN

VITRO

Early studies of the characteristics of the activation and incorporation of fatty acids into triglyceride by liver microsomes indicated a requirement for a soluble hepatic protein fraction (Hubscher et ul., 1967; Manley et al., 1974). A similar requirement for a soluble fraction of liver homogenates in addition to microsomes was found for cholesterol biosynthesis (Scallen et al., 1974; Dempsey, 1975). Several studies have suggested that L-FABP is the protein responsible for these earlier observations and that it stimulates the activities of several enzymes involved in fatty acid esterification and oxidation as well as in cholesterol biosynthesis and utilization (Table VI). Not all the observations of a stimulatory effect of L-FABP on enzyme activities have been uniformly reproduced, e.g., Suzue and Marcel (1975) were unable to document stimulation of microsomal acyl-CoA synthetase by L-FABP. Wu-Rideout et al. (1976), although finding a stimulatory effect of L-FABP on microsomal acyl-CoA synthesis, noted an inhibitory effect of the protein on the mitochondrial enzyme. These workers thus concluded that L-FABP might preferentially direct the metabolism of long chain fatty acids toward esterification rather than oxidation. Noy and Zakim (1989, using an assay in which the fatty acid substrate is incorporated in unilamellar liposomes, have found that L-FABP produces a slight inhibition of the microsomal activating enzyme. Others have also noted that the stimulatory effect of L-FABP on various enzymes is highly concentration dependent, being marked at concentrations of the protein less than 70 p M and disappearing at concentrations above 140 @4 (Burnett et al., 1979). These findings raise important questions regarding the extent to which various in vitro assays approximate conditions that exist in vivo. The mechanism underlying L-FABP stimulation of membrane-bound enzymes catalyzing fatty acid esterification is unclear, although several hypotheses exist. L-FABP may facilitate the diffusion of fatty acids or fatty acyl-CoA monomers through the aqueous medium to membranebound enzymes thus helping to maintain the concentration of these poorly

T H E CELLULAR FATTY ACID BINDING PROTEINS

167

TABLE VI EFFECTOF FABP ON ENZYME ACTIVITIES IN VITRO’ Enzyme activity Microsomal enzymes Long chain acyl-CoA synthetase

Effect of FABP

+ + + 0

-

Acyl-CoA:glycerol 3-phosphate acyltransferase Phosphatidate phosphohydrolase Diacylglycerol acyltransferase Stearoyl-CoA desaturase Ac yl-CoA:cholesterol ac yltransferase HMG-CoA reductase Methyl sterol oxidase Sterol A5-dehydrogenase Sterol A’-reductase 12a-H ydroxylase 3-Ketosteroid reductase Mitochondria1 enzymes Long chain acyl-CoA synthetase

Adenine nucleotide transporter Soluble enzymes Acetyl-CoA carboxylase

Reference Ockner and Manning ( 1976Ih Burnett ef ul. (1979) Wu-Rideout ef ul. (1976) Suzue and Marcel (1975) Noy and Zakim ( 1985)

+ + + + + + + -

Mishkin and Turcotte (1947b) Jamdar (1979) Burnett et ul. (1979) Roncari and Mack (1981) O’Doherty and Kuksis (1975) lritani ef ul. (1980) Catala (1986) Grinstead ef ul. (1983)

+ + + + + + + + + + 0 + +

Daum and Dempsey ( 1980)’ Grinstead ef a / . (1983) Scallen ef ul. (1985a) Dempsey (1975)’ Grinstead et ul. (1983) Billheimer and Gaylor (1980) Grinstead et ul. (1983) Song and Dempsey ( I97 I Ishibashi and Bloch (1981)’ Dempsey (1975)’ Scallen ef ul. (l985a) Grabowski et ul. (1976)’ Grinstead ef ul. (1983)

+ + +

Burnett ef a / . (1979) Wu-Rideout er ul. (1976) Barbour and Chan ( 1979) Lunzer et a / . (1977)

“Most of the data are for liver enzymes and L-FABP of varying degrees of purity and endogenous fatty acid content. + , Activity stimulated: - , activity inhibited; 0. no effect. hData for intestinal microsomes and a mixture of L-FABP and I-FABP. ‘Data for SCP/L-FABP.

water-soluble compounds at an optimum close to the active sites of the enzymes. Such a nonspecific “solubilizer” function for L-FABP is borne out by qualitatively similar effects of lecithin (Ockner and Manning, 1976) and albumin (Burnett et a / . , 1979; Jamdar, 1979) in several enzyme assays. On the other hand, other studies have found that, on a molar basis, albumin

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is either ineffective (Ockner and Manning, 1976; Wu-Rideout et al., 1976) or substantially less effective (Mishkin and Turcotte, 1974b; O’Doherty and Kuksis, 1975) than L-FABP in the stimulation of microsomal enzymes, thus lending weight to the argument that the effect of L-FABP may be specific to this protein and may be dependent on its direct interaction with membrane-bound enzymes. This contention is also supported by the finding that SCP/L-FABP binds in a saturable manner to proteins on microsoma1 membranes (Burton and Bloch, 1985). Jamdar (1975) presented good evidence for the mechanism of L-FABP stimulation of microsomal enzymes being closely related to the ability of this protein to protect the integrity of microsomal vesicles against the disruptive effects of long chain acyl-CoA esters. In support of this, the stimulation of acetyl-CoA carboxylase (Lunzer et al., 1977), hydroxymethylglutaryl-CoA reductase, methyl sterol oxidase, acyl-CoA: cholesterol acyltransferase (Grinstead et al., 19831, steroyl-CoA desaturase (Catala, 1986), and the mitochondria1 adenine nucleotide transporter (Barbour and Chan, 1979) by L-FABP has been attributed to the ability of the protein to sequester long chain acyl-CoA esters or fatty acids with consequent attenuation of their inhibitory effects upon these enzymes. L-FABP has also been found to stimulate several enzymes in the pathway of cholesterol and bile acid biosynthesis (Table VI), although at least in some instances, this remains controversial (Scallen et al., 1985a; Lidstrom-Olsson and Wikvall, 1986). Scallen et al. (1985a) have suggested that the activating effect of L-FABP on enzymes catalyzing the conversion of lanosterol to cholesterol may be a detergent-induced artifact, and that SCP, rather than L-FABP is solely responsible for soluble protein activation of the final steps of cholesterol biosynthesis.

B. PROTECTIVE FUNCTION The protective effect of L-FABP on several enzymatic processes that are inhibited by long chain acyl-CoA may represent specific examples of a much broader general role of the FABP as specialized intracellular “buffers.” Long chain fatty acids and their acyl-CoA esters may exert profoundly disruptive effects upon many aspects of cellular membrane integrity as well as modulatory effects upon a variety of organelle and enzyme functions (Brenner, 1984; Powell er al., 1985; Hoover et al., 1981; Rottenberg and Hashimoto, 1986; Hwang, 1986; Murakami and Routtenberg, 1985). Thus, the FABP may be important in limiting the amounts of unbound long chain fatty acids and their CoA esters over a wide range of concentrations of these substances, serving to protect many aspects of cellular function. In support of this concept is the finding that brain synaptosomal sodium-dependentamino acid transport is inhibited by long

THE CELLULAR FATTY ACID BINDING PROTEINS

I69

chain unsaturated fatty acids via a mechanism that may involve alterations in membrane fluidity (Rhoads et a / . , 1983). This inhibition is prevented and reversed by L-FABP as well as by an FABP partially purified from brain that appears to be identical with M-FABP (Bass et al., 1984a; Bass and Manning, 1986).A protective function of L-FABP may also be effected through the high affinity of the protein for heme. The latter strongly inhibits hydroxymethylglutaryl-CoA reductase, and this inhibition is prevented by L-FABP (Grinstead et al., 1983). Indeed, the impaired regulation of cholesterol and fatty acid biosynthesis observed in Morris hepatomas may reflect, at least in part, a lack of the “buffering” effect of L-FABP, the content of which is very low in this tumor line (Mishkin et al., 1977).

C. FABP I N

THE

TRANSPORT A N D UTILIZATIONOF FATTYACIDS

I . Fatty Acid Uptake In recent years, much attention has been focused on the mechanisms whereby fatty acids dissociate from albumin and are subsequently translocated into the cell interior. Some studies have suggested the presence of an albumin receptor on the hepatocyte surface that might serve to catalyze the rapid dissociation of fatty acids from albumin, thus facilitating the generation of an unbound fraction available for uptake (Weisiger et al., 1981; Ockner et a / . , 1983). However, more recent work has found that the rate of uptake of long chain fatty acids by hepatocytes is readily accounted for by the free fraction of fatty acids generated by spontaneous dissociation from albumin (Weisiger and Ma, 1987). The traditional view of cellular long chain fatty acid uptake occurring by means of passive diffusion across cell membranes (Spector and Fletcher, 1978) has also been challenged by the findings that the initial uptake of fatty acids into adipocytes (Abumrad et al., 1981) and hepatocytes (Stremmel et a/., 1985b; Stremmel and Berk, 1986) displays characteristics of a saturable, carriermediated process. Membrane carriers for long chain fatty acids have previously been well characterized in prokaryotic organisms (Black et a/., 1985) and recent work suggests that membrane carriers for long chain fatty acids may also exist in eukaryotic cells. In hepatocytes, the saturable initial uptake of fatty acids has been found to be energy and sodium dependent and inhibited up to 65% by an antibody to LPM-FABP (Stremmel et al., 1986; Stremmel and Theilmann, 1986). Stremmel (1986) has also provided strong evidence in support of the process of fatty acid translocation into liver plasma membrane vesicles being dependent, at least in part, on a potential-sensitive sodiudfatty acid cotransport system. In such a system, the energy provided by the extracellular to intracellular sodium gradient might be required to overcome the intracellular to extracellular negative-potential gradient that would oppose the movement of ionized

I70

NATHAN M. BASS

free fatty acids into the cell interior. However, Abumrad et al. (1984) have found, in contrast to the findings in hepatocytes, that the saturable uptake of fatty acids by adipocytes is neither sodium nor adenosine triphosphate (ATP) dependent. Saturable uptake of long chain fatty acids by the intestine has also been observed (Ockner et af., 1972b; Hollander et al., 1984), but in these studies, saturation of uptake may have reflected processes other than transport across the brush border membrane including the effects of the intestinal unstirred water layer and saturation of binding sites on the cytosolic FABP (Hollander et al., 1984). A role for cytosolic L-FABP in the uptake of fatty acids has also been suggested on the basis of the findings that L-FABP is increased in the livers of both female and clofibrate-treated rats compared to untreated male animals (Section VI), and that the uptake of fatty acids by isolated perfused livers of female (Kushlan et al., 1981)and clofibrate-treated rats (Renaud et af., 1978) is proportionately higher as well. However, in these studies, increased uptake rates could have reflected increased metabolic utilization of the fatty acids rather than true initial rates of movement into the cell. Indeed, substantial increases in fatty acid oxidation are well documented in clofibrate-treated animals (Cohen and Grasso, 1981; Ide and Sugano, 1983), whereas rates of both hepatic oxidation and triglyceride biosynthesis are increased in female rats (Kushlan et al., 1981; Ockner et al., 1979, 1980). Fatty acids are efficiently transferred from liposomes to L-FABP (Brecher et af., 1984). Furthermore, dissociation of pyrene fatty acid derivatives from lipid bilayers in the aqueous phase appears to be rate limiting to their overall movement between phospholipid vesicles (Doody et af., 1980). Thus, it is conceivable that the cytoplasmic FABP may be important in the transfer of fatty acids from the cell membrane to the cell interior by facilitating their dissociation from the cytoplasmic face of cell membranes. However, studies with anthroyloxy fatty acid analogs-which may be better models of long chain fatty acids than pyrene derivatives-have found that transbilayer flip-flop rather than desorption into the aqueous phase is rate limiting to overall transbilayer movement (Storch and Kleinfeld, 1986). If this is indeed the case, then the proposed carrier function of LPM-FABP may also overcome the rate-limiting step of transbilayer movement of fatty acids into the cell. On the other hand, after fatty acids have entered the cell, their binding to cytosolic FABP may limit their outward efflux and in this way influence their net uptake (Goresky et al., 1978). Recent studies have used the fluorescent fatty acid derivative 12N-methyl-7-nitrobenzo-2-oxaI ,3-diazoamino stearate (1 2-NBD-stearate), which binds tightly and almost exclusively to L-FABP in liver cytosol, to further probe the role of L-FABP in hepatic fatty acid uptake (Bass et al., 1986). Perfusion of livers via the portal vein with 12-NBD-stearate

T H E CELLULAR FATTY ACID BINDING PROTEINS

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FIG.4. lmmunoperoxidase staining the L-FABP in male rat liver. L-FABP was localized in paraformaldehyde-fixed frozen sections of 60-day-old male rat liver using specific antiL-FABP rabbit antiserum and the indirect immunoperoxidase method. Tissue sections were counterstained with Methyl Green. The photomicrograph shows positive staining for LFABP in hepatocytes close to the portal tracts (P), and negligible staining in hepatocytes surrounding the central vein (C). No staining of hepatocytes was seen in control sections incubated with nonimmune rabbit serum. x 100.

bound to albumin led to uptake that was most marked in cells in the periportal areas of the hepatic lobule, in which L-FABP is predominantly expressed (Fig. 4). However, reversal of perfusion via the hepatic veins resulted in predominant uptake of the fluorescent fatty acid by hepatocytes in the central area of the lobule in which L-FABP is least expressed. These findings suggest that the expression of L-FABP in hepatocytes is more likely to be governed by the extent to which the cells are exposed to fatty acids rather than that L-FABP is a major determinant of fatty acid translocation into hepatocytes. 2 . Intracellular Transport and Utilization of Fatty Acids Following association of fatty acids with FABP within the cell, their diffusion to enzymes responsible for their metabolic utilization must next

I72

NATHAN M . BASS

be considered. Tipping and Ketterer (1978, 1981) have constructed an elegant theoretical model of the intracellular transport of small lipophilic molecules, and, on the basis of this model, have proposed that the FABP can enhance the rate of intracellular fatty acid transport by at least an order of magnitude. This could contribute substantially to the overall efficiency of the hepatocyte in metabolizing fatty acids. Noy et al. (1986) have recently presented arguments for a different model of hepatic fatty acid transport in which the diffusion of fatty acids occurs via the cell water. However, Noy et al. (1986) assumed values for L-FABP abundance in liver cells two orders of magnitude lower than the likely minimum concentration of this protein in the cytosol (about 0.1-0.2 mM; e.g., see Tipping and Ketterer, 1978; Burnett et al., 1979), an error that casts serious doubt upon the validity of their model. Much of the support for a role for the FABP in promoting the cellular utilization of fatty acids has come from the experiments discussed earlier (Section VILA) examining FABP effects on isolated organelles. Additional, albeit far more circumstantial, evidence for this function of the FABP has also been obtained from experiments using intact cells. Several studies have demonstrated that flavispidic acid, a competitive inhibitor of fatty acid binding to L-FABP, inhibits the esterification and oxidation of fatty acids in the intact cells of liver and intestine (Burnett et al., 1979; Ockner and Manning, 1976; Mishkin et al., 1975). These findings could, however, reflect either direct inhibition by flavispidic acid of the enzymes responsible for fatty acid activation and utilization (Burnett et al., 1979) or displacement of fatty acids from FABP, thus preventing FABP-mediated donation of fatty acids and fatty acyl-CoA to the enzymes. Similarly, the finding that partially purified FABP prevented the inhibition of mitochondria1 and microsomal long chain acyl-CoA synthetase by flavispidic acid (Burnett et al., 1979) is open to several interpretations, including binding of the inhibitor by FABP. In studies utilizing everted gut sacs, Ockner ef al. (1972b) found that although palmitate uptake equaled or exceeded that of linoleate. palmitate esterification was significantly less rapid. Since the apparent affinity of L-FABP is greater for long chain unsaturated than for saturated fatty acids (Ockner et al., 1972a; Mishkin ef al., 1972), it is conceivable that the differences in the rates of intestinal esterification of palmitate and oleate reflect their different affinities for L-FABP. Indeed, the concentration of L-FABP in the liver is increased in several situations in which hepatic trigyceride biosynthesis is increased, including feeding rats high fat diets (Table V; Brindley, 19781, alcohol (Pignon et al., 1986), cholestyramine (Kempen ef al., 1983). and also in the livers of female or estrogen-treated male rats (Ockner ef al., 1979, 1980). Ockner et al. (1980) found a strong

THE CELLULAR FATTY ACID BINDING PROTEINS

173

correlation between the hepatic concentration of L-FABP and triglyceride biosynthesis in hepatocytes isolated from normal adult rats and also from rats that had undergone gonadectomy with or without sex steroid hormone supplementation (Fig. 5 ) . The additional finding that several microsomal enzymes involved in triglyceride biosynthesis showed no sex difference in specific activities suggested that L-FABP might be primarily responsible for sex steroid hormone-dependent differences in triglyceride biosynthesis (Ockner et al., 1979). However, others have found increased activity of phosphatidate phosphohydrolase (Savolainen et al., 198 1) and higher intrinsic microsomal triglyceride biosynthetic activity (Soler-Argilaga et ul., 1978) in female rat livers. Furthermore, immature rat hepatocytes, although having lower concentrations of L-FABP than those found in adults, are capable of relatively high rates of triglyceride biosynthesis (Fig. 5 ) and also contain higher specific activities of several microsomal enzymes involved in triglyceride biosynthesis than those found in the livers of mature

T

7

bO?C 1

/ r=0.94

p<.oo1

I

6

1

8

I

10

FABP (nmol I4C-oleate bound) FIG.5 . Relationship between triglyceride biosynthesis in isolated hepatocytes and cytosolic L-FABP concentration. Group mean values for incorporation of [14Cloleateinto triglycerides by hepatocyte suspensions are plotted as a function of the corresponding mean values for L-FABP concentration in hepatic cytosol. The linear regression was calculated for 60-day-old animals only. FABP was measured by ['4C]oleate binding assay. Symbols: 30. 60. ages in days; F, female; M. male; E, estradiol treated; T, testosterone treated; C. castrated. Reproduced from Ockner ct a / . (1980) by copyright permission of the American Society for Clinical Investigation and the authors.

174

NATHAN M. BASS

animals (Ockner et al., 1980). Thus, the relative contributions of L-FABP and triglyceride biosynthetic enzyme activity as well as other factors (Pikkukangas et al., 1982) toward the ultimate rate of fatty acid incorporation into triglyceride are impossible to dissect at present. Furthermore, it is unknown whether the sex difference in L-FABP is causal or coincidental to sex differences in triglyceride biosynthesis, and whether sex-dependent modulation of L-FABP (see Section VI,C) is affected by sex steroid hormones directly or via alterations in hepatic fatty acid flux determined by sex-dependent factors. An increase in hepatic L-FABP is not only associated with states of chronically increased triglyceride biosynthesis. This is notably the case in the livers of animals treated with peroxisome proliferators (Section VI,D) in which triglyceride biosynthesis is considerably reduced, while phospholipid synthesis and fatty acid oxidation are increased (Ide and Sugano, 1983). Following the administration of a variety of peroxisomeproliferating agents, a strong correlation between the induction of peroxisomal p oxidation and L-FABP in liver was found (Kawashima et al., 1983) and led to speculation regarding a role for the protein in providing fatty acids for peroxisomal p oxidation. Indeed, Appelkvist and Dallner (1980) have presented experimental data suggesting a role for L-FABP as a carrier involved in the transfer of long chain acyl-CoA derivatives through the peroxisomal membrane. These studies, however, used whole cytosol as a source of L-FABP, and require confirmation using the pure protein. Clearly, the putative function of the FABP as intracellular carrier proteins that facilitate the intermembranous diffusion of fatty acids and fatty acyl-CoA to their sites of metabolic utilization (mitochondria and endoplasmic reticulum) requires further direct experimental evaluation. The transfer of fatty acids from liposomes (Brecher et al., 1984; Reers et al., 1984) and lipid droplets (Scallen et al., 1985a) to L-FABP has been demonstrated. Furthermore, L-FABP appears to increase the rate of transfer of long chain fatty acids from microsomes and mitochondria to liposomes (Catala and Avanzati, 1983). However, L-FABP was found to retard the transfer of palmitate from liposomes to microsomal palmitoyl-CoA synthetase (Noy and Zakim, 1985). These findings, collectively, fully support the possible protective function of L-FABP discussed earlier (Section VII,B), but do not permit any definite conclusion to be drawn regarding a fatty acid carrier-donor role for this protein. 3. Structure-Dependent Specialization of FABP Function

In distinct contrast to L-FABP, M-FABP readily donates anthroyloxy palmitate to unilamellar liposomes (Reers et al., 1984), suggesting that these two structurally different FABPs may be functionally specialized as well. FABP purified from mammary gland is similar or identical to M-

THE CELLULAR FATTY ACID BINDING PROTEINS

175

FABP (Jones et al., 1986),and does not affect the activities of microsomal acyl-CoA synthetase, phosphatidate phosphohydrolase, or diacylglycerol acyltransferase (Whetstone et al., 1986).On the other hand, M-FABP has been shown to transfer long chain fatty acids to isolated mitochondria where they subsequently undergo f3 oxidation (Fournier et al., 1978; Glatz et al., 1985). The tissue distribution of M-FABP is also compatible with a role for this protein predominantly in connection with fatty acid oxidation (Bass and Manning, 1986). Based on the observations that M-FABP undergoes a concentration-dependent transition between different states of self-aggregation, and that self-aggregates of differing molecular weight have demonstrably different abilities to bind and to provide fatty acids to mitochondria for f3 oxidation, Fournier and Rahim (1985) have proposed that M-FABP, through its self-aggregating properties, regulates the rate of heart mitochondria1 f3 oxidation, possibly in concert with the cardiac contractile cycle. Others have not, however, been able to reproduce the self-aggregating phenomenon upon which this entire premise rests (Jagschies et al., 1985; Offner et al., 1986). Differences in the function of IFABP and L-FABP are also suggested by the differences in the response of these two proteins to various modulatory influences (Section VI), but direct evidence for their functional specialization is lacking. 4. The Cellular Transport of Other Small Molecules

Many amphipathic small molecules apart from fatty acids bind to LFABP, and the question arises as to whether or not this protein serves a broad function in the intracellular transport and/or sequestration of amphipathic small molecules. Although this is indeed feasible, three points argue for a function for L-FABP related primarily to the cellular transport and utilization of fatty acids. First, the flux of long chain fatty acids through the liver dwarfs that of other amphipathic small molecules such as bilirubin, bile acids, or xenobiotics (Burnett et al., 1979). Second, apart from heme, the binding affinityof small molecules to L-FABP are less than the affinity of long chain fatty acids for this protein (Section V,B). Third, the binding of long chain fatty acids and fatty acyl-CoA to cytosol proteins shows a high degree of specificity for L-FABP (Ockner et al., 1972a, 1982; Mishkin et al., 1972; Mishkin and Turcotte, 1974a; Kawashima et al., 1984), whereas most other amphipathic small molecules are bound with equal or higher affinity to the abundant family of glutathione S-transferases (Ketterer et al., 1978; Ketley et al., 1975) as well as other cytosolic binding proteins (Sugiyama et al., 1982b). Furthermore, although studies using isolated liver perfusion have suggested that L-FABP may reduce the efflux of long chain fatty acids from the liver (Goresky et al., 1978), similar experiments have concluded that L-FABP does not limit the hepatic efflux of bilirubin (Thielman et al., 1984) but rather that this is achieved by the

176

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cytosolic glutathione S-transferases (Wolkoff et a f . , 1979). Several cytosolic proteins bind heme with affinities equal to or greater than that of L-FABP (Tipping et al., 1976; Muller-Eberhard and Vincent, 1985), and a role for a soluble glutathione S-transferase rather than L-FABP has been implicated in the transport of heme from mitochondria to hemoproteins (Senjo et al., 1985). 5 . Summary of Current Concepts of FABP Function In conclusion, currently available data have strongly suggested a role for the 40-kDa LPM-FABP as a specific cell membrane carrier in the transport of long chain fatty acids into the cells of the liver and possibly other tissues as well. The 14- to 15-kDa cytosolic FABPs may function as intracellular acceptors and carriers of long chain fatty acids and their CoA esters, maintaining the ability of the tissues, particularly liver, intestine, heart, and skeletal muscle, to utilize fatty acids over wide and acutely varying ranges of flux. Data on the turnover of L-FABP and its response to acute changes in fatty acid flux and utilization such as occur in starvation and refeeding do not support a role for this protein in modulating rapidly changing events in fatty acid transport and metabolism. However, a diverse body of evidence has lent indirect support for a role for the FABPs as determinants, at least in part, of the intrinsic ability of cells to modulate rates of fatty acid utilization over longer periods of time. There is at present no conclusive evidence that the various FABPs may preferentially direct fatty acids toward utilization by either esterification or oxidative pathways, although most data support a role for M-FABP in providing fatty acids for mitochondria1 p oxidation. L-FABP may also be important in the cellular economy of fatty acids, maintaining a specific pool of essential fatty acids for the purposes of phospholipid, prostaglandin, and leukotriene synthesis. Furthermore, by preventing fluctuations in the concentration of unbound fatty acids, fatty acyl-CoA esters, and other metabolites, and by removing these compounds from cell membranes, the FABP may serve an essential role in protecting many aspects of cell function. Indeed, it is quite feasible that the increments in cytosolic FABP concentrations observed in several diverse states of chronically increased cellular fatty acid flux and utilization represent an adaptive response of primarily protective significance. Finally, although there is considerable evidence supporting the multitude of functions of the cytosolic FABP outlined above, much of this evidence is circumstantial, and many details, in particular those regarding the putative transport function of these abundant proteins, their interaction with cell membranes and enzymes, and the mechanisms and significance of their regulation, remain to be fully elucidated. Questions pertaining to the possible functional specialization of the different structural types of cytosolic FABP will undoubtedly also be addressed by future research.

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ACKNOWLEDGMENTS The author acknowledges with deep gratitude the invaluable advice and insight of Robert K. Ockner and the technical support of Joan A. Manning. The author also thanks Jeffrey I. Gordon and Richard A. Weisiger for helpful discussions, Mary Barker for preparing the photomicrographs in Fig. 4, and Michael Karasik and Diana Fedorchak for editorial assistance in preparing the manuscript. This work was supported in part by Research Grant AM-32926 and Liver Core Center Grant AM-26743 from the National Institutes of Health and the American Gastroenterological AssociatiodJanssen Pharmaceutica Research Scholar Award. REFERENCES Abumrad, N. A., Perkin, R. C., Park. J. H., and Park, C. R. (1981). J . Biol. Chem. 256, 9183-9191. Abumrad, N. A., Park, J. H., and Park, C. R. (1984). J . Biol. Chem. 259, 8945-8953. Alpers, D. H., Strauss, A. W., Ockner, R. K., and Bass, N. M.(1984). Proc. Natl. Acad. Sci. U.S.A. 81, 313-317. Appelkvist, E. L., and Dallner, G. (1980). Biochim. Biophys. Acra 617, 156-160. Barbour, R. L., and Chan, S. H. P. (1979). Biochem. Biophys. Res. Commun. 89, 11681177. Bass, N. M. (1985). Chem. Phys. Lipids 38, 95-114. Bass, N. M., and Manning, J. A. (1986). Biochem. Biophys. Res. Commun. 137, 929-935. Bass, N. M., and Ockner, R. K. (1987). In “Proceedings of the IX International Symposium on Drugs Affecting Lipid Metabolism” (W.L. Holmes, D. Kritchevsky. and R. Paoletti. eds.). Springer-Verlag, Heidelberg, in press. Bass, N. M.. Raghupathy, E., Rhoads. D. E., Manning, J. A., and Ockner, R. K. (1984a). Biochemistry 23,6539-6544. Bass. N. M., Manning, J. A., Weisiger, R. A., and Ockner, R. K. (1984b). Hepatology 4, 1016. Bass, N. M., Manning, J . A., Ockner, R. K., Gordon, J. I . , Seetharam, S., and Alpers. D. H. (1985a). J . Biol. Chem. 260, 1432-1436. Bass, N. M., Manning, J. A., and Ockner, R. K. (1985b). J . Biol. Chem. 260, 9603-9607. Bass, N. M., Barker, M. E., Jones, A. L., and Ockner, R. K. (1985~).Hepatology 5 , 1011. Bass, N. M., Manning. J. A., and Ockner, R. K. (1986). Gastroenterology 90, 1710. Basu. S. P., Narasinga Rao, S., and Hartsuck, J. A. (1978). Biochim. Biophys. Acra 533, 66-73. Bernlohr, D. A., Angus, C. W., Lane, M. D., Bolanowski, M. A., and Kelly, Jr., T. J. (1984). Proc. Narl. Acad. Sci. U.S.A. 81, 5468-5472. Bernlohr, D. A., Doering, T. L.,Kelly, T. J., and Lane, M. D. (1985). Biochem. Biophys. Res. Commun. 132, 850-855. Billheimer, J. T., and Gaylor, J. L. (1980). J . Biol. Chem. 255, 8128-8135. Black, P. N., Kianian, S. F., DiRusso, C. C., and Nunn, W. D. (1985). J. Biol. Chem. 260, 1780-1789. Brandes. R.. and Arad, R. (1983). Biochim. Biophys. Acta 750, 334-339. Brecher, P., Saouaf, R., Sugarman, J. M., Eisenberg, D., and LaRosa, K. (1984). J . Biol. Chem. 259, 13395-13401. Brecher, P., Saouaf, R., Grice, W., and Apstein, C. S. (1986). Clin. Res. 34, 707A. Brenner, R. R. (1984). Prog. Lipid Res. 23, 69-96. Brindley, D. N. (1978). In “Regulation of Fatty Acid and Glycerolipid Metabolism” (R. Dils and J. Knudsen, eds.), Vol. 46, pp. 31-40. Pergamon, New York. Burnett, D. A., Lysenko, N., Manning, J. A.. and Ockner, R. K. (1979). Gastroenterology 77, 241-249.

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cell membranes (McCormack and Brecher, 1987). binding of acyl-CoA product, and protecting acyl-CoA from hydrolysis (Burrier et a/., 1987). Recent work has also shown that L-FABP is identical to a protein abundantly expressed in hepatocytes undergoing mitosis in regenerating and carcinogen-treated livers (Bassuk et d..1987)and that the FABPare structurally and immunologically related to growth inhibitor polypeptides derived from mammary gland (Bohmer et a / . , 1987a) and fibroblasts (Bohmer et d.,1987b). These exciting new findings raise the important possibility that the FABP may play a role, conceivably via their ligand-binding properties, in the regulation of cell growth and proliferation. REFERENCES TO NOTEADDEDIN PROOF Bassuk, J. A., Tsichlis, P. N., and Sorof, S. (1987). Proc. Nut/. Acud. Sci. U . S . A . 84,7547755 I . Bohrner. F.-D., Krafts, R., Otto, A., Wernstedt, C.. Hellman, U.. Kurtz, A., Muller, T.. Rohde, K.. Etzold, G., Lehmann, W., Langen, P., Heldin. C.-H., and Grosse, R. (1987a). J . Biol. Chem. 262. 15137-15143. Bohmer. F.-D., Sun, Q., Pepperle, M., Muller, T.. Eriksson. U., Wang, J. L.. and Grosse, R. (1987b). Biochem. Biophys. Res. Commun. 148, 1425-1431. Burrier, R. E.. Manson. C. R., and Brecher. P. (1987). Biochim. Biophys. Actu 919, 221230. Crisman. T. S., Claffey, K. P., Saouaf, R., Hanspal, J., and Brecher. P. (1987). J . M o l . CeII Curdiol. 19, 4 2 3 4 3 I. Dutta-Roy. A. K., Gopalswamy, N.. and Trulzsch, D. V. (1987). Eur. J . Biochem. 162, 6 15-619. Fujii. S., Kawaguchi, H., and Yasuda, H. (1987a). J . Biochem. 101, 679-684. Fujii. S., Kawaguchi, H., and Yasuda, H. (1987b). Arch. Biochem. Biophys. 254, 552-558. Gibson. B. W., Yu, Z., Aberth, W., Burlingame, A. L., and Bass, N. M. (1987). In "Proceedings of the 35th ASMS Conference on Mass Spectrometry and Allied Topics," pp. 944-945. ASMS, Denver, Colorado. Heuckeroth, R. 0..Birkenmeier, E. H., Levin, M. S., and Gordon, J. 1. (1987). J. Biol. Chetn. 262, 9709-9717. Lowe, J. B., Sacchettini, J. C., Laposata, M., McQuillan, J. J.. and Gordon, J. I. (1987). J . Biol. Chem. 262, 5931-5937. McCormack. M.. and Brecher. P. (1987). Biochem. J . 244, 717-723. Sacchettini, J. C., Meininger, T. A., Lowe, J. B.. Gordon, J . I., and Banaszak, L. J . (1987). J . B i d . Chem. 262. 5428-5430. Sweetser, D. A., Heuckeroth, R. 0.. and Gordon. J. 1. (1987a). Ann. Rev. Nirtr. 7 , 337359. Sweetser. D. A., Birkenpeier. E. H., Klisak, I. J., Zollman, S.. Sparkes, R. S.. Mohandas. T.. Lusis, A. J., and Gordon, J. I. (1987b). J . Biol. Chem. 262, 16060-16071. Veerkamp. J. H., and Paulussen, R. J. A. (1987). Biochem. Soc. Truns. 15, 331-336. Wilkinson, T. C. I., and Wilton, D. C. (1987). Biochern. J . 247, 485488.