Fatty acid trafficking in the adipocyte

seminars in C E L L & D E V E L OP M E N T A L B I OL OG Y , Vol 10, 1999: pp. 43]49 Article No. scdb.1998.0271, available online at http:rrwww.idealibrary.com on

Fatty acid trafficking in the adipocyte David A. Bernlohr U , Natalie Ribarik Coe U and Vince J. LiCata†

The insolubility of fatty acids in cellular environments requires that specific trafficking mechanisms be developed to vectorally orient and deliver lipids for cellular needs. The roles of putative membrane bound fatty acid transporters and soluble carrier proteins are discussed in terms of mechanisms of fatty acid trafficking. The numerous roles for fatty acids as an energy source, as structural elements for membrane synthesis, as bioregulators and as prohormones with the potential to regulate gene expression, are discussed in terms of the necessity to regulate their intracellular location and concentration.

but also fatty acid signalling. Several proteins implicated in controlling fatty acid flux, such as fatty acid transporters and the intracellular lipid binding proteins will be introduced and the more recent interpretations of their prospective roles in fatty acid utilization, storage andror trafficking will be discussed in detail. The role of fatty acids as modulators of cellular function, and as possible trafficking agents will be considered.

Key words: fatty acid r trafficking r lipid-binding proteins r transporters r metabolic control

Adipocyte plasma membrane fatty acid transporters

Q1999 Academic Press

Fatty acids destined for adipose cells circulate in plasma in the form of triglyceride bound to lipoprotein particles. Hydrolysis of lipoprotein triglycerides by lipoprotein lipase liberates fatty acids which are bound by serum albumin. Albumin-bound fatty acids traverse the endothelial cell layer in an ill-defined manner and may interact directly with the adipocyte plasma membrane to stimulate fatty acid uptake,1 although to date an albumin receptor has yet to be unequivocally identified. The actual mechanism of transmembrane fatty acid flux is controversial. Fatty acids may enter fat cells by means of diffusional fatty acid flip-flop 2 ] 5 or with the aid of one or more plasma membrane transport proteins.6 ] 10 Given the high concentration of serum albumin in the extracellular space and its binding constants for fatty acids, the free, unbound fatty acid concentration is in the nanomolar range,11 hence the consideration of a protein-mediated mechanism for fatty acid influx into the adipocyte. Although the necessity of proteinmediated transport remains controversial, a few promising candidates have been isolated and preliminarily characterized. The ability of fatty acids to cross the adipocyte plasma membrane is critical to not only the maintenance and mobilization of stored energy reserves but

Overview FATTY ACID TRAFFICKING IN adipose tissue is a complex and dynamic process that affects many aspects of cellular function ŽFigure 1.. Several features of lipid metabolism need to be considered when examining the flux of fatty acids into, within and from the fat cell. This short review will examine the role of fatty acids as both an energy source and as a metabolic regulator, acting in partnership with transcriptional activators such as peroxisome proliferator-activated receptors ŽPPARs. to regulate gene expression. Adipose proteins, by controlling the concentration, localization and availability of fatty acids, are therefore intimately involved in not only energy metabolism, U

From the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1479 Gortner Ave, St. Paul, MN 55108, USA and †Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803, USA Q1999 Academic Press 1084-9521r 99r 010043q 07 $30.00r 0

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D. A. Bernlohr et al

Figure 1. The multifaceted role of fatty acids in adipose tissue. Fatty acids or metabolites are used for cellular processes as diverse as Ž1. transcriptional control; Ž2. membrane synthesis; Ž3. regulators of cellular metabolism; and Ž4. energy storage in the triglyceride droplet. FA, fatty acids; PPAR, peroxisome proliferator activated receptor; RXR, retinoid X receptor; UCP, uncoupling protein. v, fatty acid andror derivative; ^, 9-cis retinoic acid.

also to the ability of the cell to respond to changes in extracellular fatty acid concentration and flux. Fatty acid binding protein, plasma membrane ŽFABPPM ., a candidate transporter identified by Berk, Stremmel and colleagues, was originally isolated directly from rat hepatocytes12 and subsequently from rat adipocytes.13 Shortly thereafter, FABPPM was shown to be identical to the previously known mitochondrial protein, aspartate aminotransferase.14 The early 1990s brought reports of three additional plasma membrane proteins with speculated involvement in fatty acid uptake. Trigatti et al 7 identified the 22-kDa 3T3-L1 adipocyte plasma membrane protein caveolin as being capable of binding a photoreactive fatty acid analogue with high affinity and as a possible transporter. Caveolin is a structural component of caveloae, also known as plasmalemmal vesicles, which form invaginations or pits in the plasma membrane of many different cell types, including adipocytes. Abumrad and colleagues have biochemically studied in detail an 88-kDa plasma membrane protein termed the fatty acid translocase ŽFAT.. In 1993, FAT was cloned from a rat fat cDNA library 12 revealing that it

was in fact the rat homologue to human CD36, a glycoprotein previously localized to platelets and mammary epithelial cells thought to facilitate the clearance of oxidized lipoprotein particles via association with an extracellular lipid binding domain.12,15,16 More recently, a protein termed fatty acid transport protein ŽFATP. was identified by Schaffer and Lodish8 from a 3T3-L1 adipocyte cDNA expression library. Unlike its predecessors, FATP was identified based on a functional uptake assay using a fluorescent fatty acid analog, not its ability to bind fatty acid analogues directly. Influx studies, utilizing either primary culture adipocytes or 3T3-L1 adipocytes, have demonstrated a positive correlation between the expression of either the fatty acid translocase ŽFAT.17 ] 19 , FATP 8,19 or FABPPM 14 and fatty acid uptake. Antibodies against FABPPM were able to reduce oleate uptake by confluent 3T3-L1 cells by approximately 50%.20 Similarly, disruption of the FATP yeast homologue in Saccharomyces cerevisiae resulted in impaired oleate uptake21 and decreased very long-chain fatty acyl CoA synthetase activity.22 It is not clear if these de44

Fatty acid trafficking in the adipocyte

scribed putative transporters work individually, synergistically, or as part of a larger complex that may involve additional extracellular or intracellular proteins. Extensive work examining the regulation of expression for these putative transporters has been carried out.23 The tissue distribution and expression level appears different for FABPPM , FAT and FATP. For example, FABPPM has been localized to liver, skeletal muscle, intestine and testes, in addition to adipose. The expression of the fatty acid translocase is highly regulated, being elevated in oxidative red skeletal muscle relative to glycolytic white fibers.24 It is also upregulated three to fourfold in heart muscle of mice maintained on a 40% fat diet when compared to littermates fed a 9% fat chow;25 FABPPM FAT and FATP are also upregulated in the adipose of Zucker homozygous fatty rats Žgenetic obesity model..19 FABPPM , FAT, FATP and caveolin are all upregulated during adipogenesis, the differentiation of fibroblastic preadipocytes to mature fat cells.20,26 ] 28 FATP in adipose tissue of normal mice is downregulated by insulin and upregulated by fasting, two conditions which are seemingly inconsistent with a role in fatty acid uptake.27 Thus, FATP may function as a bi-directional transporter which would be expressed in tissues that explicitly take up fatty acids, such as skeletal muscle, as well as adipose which is capable of both fatty acid uptake and efflux. It is important to recognize that additional factors, specific to tissue type, may be needed to facilitate efficient and regulated fatty acid transport at the plasma membrane level. A second group of such proteins implicated in intracellular fatty acid trafficking are the lipid-binding proteins.

approximately 99:1 ŽRibarik Coe et al, submitted manuscript.. ALBP is expressed only in adipose tissue,31,32 whereas KLBP is found in a variety of tissues.29,33 Due to their intracellular abundance and ability to bind a wide variety of lipids, LBPs are generally acknowledged to be key players in intracellular fatty acid trafficking even though their role remains very much unresolved. The two primary competing physiological models for LBP function are: Ž1. that they serve a passive ‘buffering’ role whereby fatty acids and other lipids remain soluble and functionally accessible in the cytosol by virtue of being bound to an LBP; and Ž2. that the LBPs serve as active chaperones facilitating specific transport of lipids within the cytosol and participating in specific targeted interactions with other intracellular partners such as membranes or other proteins. These two roles are not mutually exclusive, and any experiment supporting one model does not necessarily exclude the other. It may very well be that LBPs serve in both active and passive capacities in intracellular lipid trafficking, and that the search for a specific function is an oversimplification. LBPs have been investigated from a number of different perspectives: they have been characterized as isolated proteins by X-ray crystallography,34 NMR35,36 and other biochemical and biophysical techniques;37,38 their genomic organization and regulation of gene expression has been studied in a large number of tissues and organisms;39 and their behavior and potential functionŽs. have been examined in cultured and primary cells and in LBP-null transgenic mice.29 These different investigational approaches each provide unique information, all of which must be integrated into any satisfactory understanding of the physiological roleŽs. of LBPs in any tissue. LBPs exhibit an extremely wide range of sequence diversity, with different members of the family ranging between 15 and 80% sequence identity.34 The two adipocyte LBPs are 55% sequence identical.38 In contrast to this tremendous range of sequence homology is the fact that all known LBPs share almost identical three-dimensional structures. All LBPs fold into 10 stranded anti-parallel b-barrels with a helixturn-helix ‘cap’ and an interior cavity where fatty acids are bound.34 Furthermore, the gene structure of all known LBPs is conserved, each having four exons and three introns. ALBP, like the majority of the LBPs, exists as a single copy gene.39 KLBP is one of the few LBPs to have multiple genome copies, although only one copy may be functional.39,40

Intracellular lipid-binding proteins Lipid-binding proteins ŽLBPs. are a family of small monomeric soluble proteins that bind fatty acids, retinoids, and other lipids. Different members of the LBP family exhibit unique patterns of tissue expression, and are expressed abundantly in tissues involved in active lipid metabolism, i.e. skeletal muscle, liver, intestine, brain, white adipose, brown adipose. LBPs have been the subject of several recent reviews.29,30 White adipose contains two different LBPs: adipocyte lipid binding protein ŽALBP or aP2. and keratinocyte lipid binding protein ŽKLBP.. ALBP has also been localized to brown adipose. The ratio of ALBP to KLBP in adipocytes isolated from normal mice is 45

D. A. Bernlohr et al

Table 1. Biochemical and biophysical properties of the two adipose lipid binding proteins Žadapted from Simpson et al 38 . Property

ALBP

KLBP

No. amino acids

131

135

Molecular weight

14,578

15,137

Tissue distribution

Adipose y1

Abundance in adipose

60 mg g

pI

9

Skin, adipose, lens, epithelium total protein

0.6 mg gy1 total protein 6.5

y1

DGunfolding

y5.3 kcal mol

y2.3 kcal moly1

Affinity Ki ŽnM. Myristate Palmitate Oleate Linoleate

506 390 215 368

1873 1087 320 499

Viewed at the level of protein structure, the LBPs vis a vis their sequence diversity and conserved fold, have essentially introduced the potential for a wide array of surface microspecificity within the same three-dimensional package. A recent comparison of the surface characteristics of the two LBPs of adipocytes showed a variety of differences between the two proteins.38 A variety of other biochemical and biophysical data, summarized in Table 1, also indicates that the sequence diversity between the two proteins results in a variety of differences in their chemistry and predicted topology. The primary question is: are these differences functionally significant? These differences will certainly cause KLBP and ALBP to function differently within the cell. Differing electrostatic contours surrounding the proteins38 will result in different rates of ligand approach to the two proteins, and in different affinities for charged ligands ŽTable 1.. Moreover, the altered folding stabilities will result in different intracellular half lives and different relative stabilization upon lipid binding. The different pIs would result in differential intracellular sorting. In general, these types of differences would be presumed to be indicative of the fact that KLBP and ALBP play very different roles within the cell: that they interact with different but non-exclusive sets of intracellular partners, and that they are likely to be compartmentalized differently within the cell. The high concentrations of LBPs in adipocytes, 1]3% of the soluble protein in adipocyte, up to 8% in the other tissues29,41 coupled with the high binding affinities for various fatty acids implies that the free, unbound fatty acid concentration within the cell is extremely low, roughly nanomolar. The signifi-

cance of this addresses the reported feedback inhibition of several metabolic enzymes by fatty acids. The activity of the hormone sensitive lipase,42 the insulin receptor tyrosine kinase43 and the acetyl CoA carboxylase 44 are all inhibited by fatty acids. While direct inhibition is an attractive model for molecular regulation, the concentration of fatty acids necessary to affect activity Žmicromolar. is approximately 1000 times higher than the likely cellular free unbound fatty acid concentration. Consequently, regulation of metabolic enzymes by such levels of fatty acids is not likely to be of physiological significance.

Trafficking of fatty acids in the adipocyte While the trafficking patterns for fatty acids have not been established in adipocytes, or in any cell type, thoughtful consideration of the likely routes suggests a number of targets ŽFigure 2.. The most commonly considered site for trafficking of fatty acids in adipocytes is from the plasma membrane transporters to the acyl CoA synthetases present on several internal membranes. Acyl CoAs in turn are readily utilized for membrane and triacylglycerol synthesis. Dietary fatty acids destined for storage must be physically moved from their site of entry at the cell surface to inner membranes in order to be converted to their acyl CoA thioesters by fatty acyl CoA synthetaseŽs.. Several classes of CoA synthetases cover the spectrum of available fatty acid substrates. Acyl CoA synthetases are classified based upon the chain length of their preferred substrates, namely short 8C, medium Ž8]12 C., long-chain Ž14]20 C., and very-long-chain acyl 46

Fatty acid trafficking in the adipocyte

Figure 2. Fatty acid trafficking routes in adipocytes. The postulated routes for fatty acid trafficking in adipocytes are presented. Lipid-binding proteins bind intracellular fatty acids and may aid in fatty acid transport to cellular locales, such as the nucleus or mitochondria, andror to enzyme partners, such as acyl CoA synthetase.

CoAs Ž20 C and greater.. Insofar as intracellular lipid binding proteins are concerned, lipid binding protein trafficking would affect only those acyl CoA synthetases which utilize long and very long chain fatty acids, the natural substrates for LBPs. A second potential site for trafficking of fatty acids is during lipolysis ŽFigure 2.. The product of lipolysis, fatty acids, are inhibitory towards the hormone sensitive lipase and must be bound by a carrier in order to efficiently carry out efflux. It is likely that trafficking of fatty acids from the droplet surface where active hormone sensitive lipase ŽHSL. resides to the plasma membrane for efflux is facilitated by LBPs. However, since a considerable fraction of lipolytically-derived fatty acids are re-esterified to their CoA esters, trafficking of newly liberated fatty acids back into the biosynthetic pathway and away from the efflux pathway is also likely to occur. Another attractive possibility is for trafficking of fatty acids or fatty acid metabolites to nuclear sites. Nuclear receptors such as the peroxisome proliferator-activated receptors associate with fatty acids and their metabolites to activate gene expression.45 Per-

oxisome proliferator activated-receptors ŽPPARs. are 50]60-kDa nuclear transcription factors which exist in multiple isoforms Ž a , d ,g .. Isoforms a and g are found in adipose tissue and activators include synthetic compounds such as WY14,643 Ž a-specific . and thiazolidinediones Žg-specific . or arachidonic acid metabolites such as 15-deoxy-D 12,14-prostaglandin J2 .46 PPARs form heterodimers with RXR a Žretinoid X receptor, 9-cis retinoic acid binding. to activate the expression of target genes containing peroxisome proliferator responsive elements ŽPPRE.. Many genes encoding proteins involved in lipid metabolism are regulated by PPAR activators: fatty acid translocase47 and FATP,48 adipocyte lipid binding proteinraP2,46 mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase ŽHMG-CoA synthase..49 Consequently, an autoregulatory positive feedback loop exists in which adipocyte fatty acid transporters andror lipid binding proteins may facilitate the trafficking of fatty acid metabolites destined to be used as co-activators, of the expression of their own genes. The targeted delivery of fatty acids to the brown fat uncoupling protein is an additional site for LBP]pro47

D. A. Bernlohr et al

tein interaction. The uncoupling protein facilitates the non- ATP- coupled dissipation of the electrochemical potential via the passage of protons across the mitochondrial membrane. Based upon the work of Garlid and colleagues,50,51 the counterion for proton transport is considered to be a fatty acid. However, utilizing UCP reconstituted in proteoliposomes, Winkler and Klingenberg 52 have put forth a model in which fatty acids provide buffering capacity for protons as they are transported across membranes by UCP, although the exact role and need of fatty acids is highly disputed.52 ] 54 Nevertheless, delivery of fatty acids to the brown fat mitochondrial outer membrane to be used for thermogenesis is an attractive hypothesis for adipocyte lipid trafficking.

5. 6. 7. 8. 9. 10.

11.

12.

Conclusion The trafficking of fatty acids within the adipocyte is a process necessary to facilitate the rapid control of metabolism in response to changes in biosynthetic availability of nutrients. Plasma membrane transporters and cytoplasmic lipid carriers are the likely agents utilized by adipocytes to facilitate such vectoral directed movement of fatty acids. Given the abundance of lipid binding proteins, their physical properties and affinity for fatty acids, it is likely that they participate in low-affinity associations with numerous intracellular binding partners. Concerted effort is now being undertaken to identify such binding partners and characterize the molecular interactions.

13.

14.

15. 16.

17.

Acknowledgements 18.

This work supported by grants from the National Institutes of Health and the National Science Foundation. 19.

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