The specific roles that the multiple FABP isotypes play in the biology of their ligands is not well understood. It is usually believed that these proteins serve a general function in solubilization and trafficking of their ligands and that they affect lipid metabolism through their transport function (3
). Support for the involvement of FABPs in fatty acid transport has been provided by reports that diffusion of these ligands both in vitro and in cells is facilitated in the presence of FABP (46
). The nonspecific nature of this activity has been emphasized by the observation that bovine serum albumin could replace FABP in establishing intracellular diffusive flux of fatty acids (29
). Other roles for FABPs have been implied by some studies. It has been suggested that I-FABP, H-FABP, and A-FABP may facilitate the dissociation of fatty acid from membranes (19
). It has also been reported that H-FABP associates with the scavenger receptor CD36 (41
), although the functional significance of this observation remains unclear. Recently, the possibility that a connection exists between the activities of FABPs and PPARs has been explored. It was reported that L-FABP associates with specific nuclear membrane proteins in the presence of long-chain fatty acids or synthetic PPAR ligands (25
). Overexpression of A-FABP and K-FABP was reported to inhibit the transcriptional activities of all three PPAR subtypes both in the absence and in the presence of exogenous ligand (18
). It was also recently shown that L-FABP interacts with PPARα and PPARγ both in the absence and in the presence of ligands and that decreased expression of L-FABP in cells results in down-regulation of the transcriptional activities of ligands for all three PPAR isotypes (49
). The mechanism by which L-FABP may exert these seemingly nonselective effects has not been clarified. We note as well that the basis of the inconsistency between the report that A-FABP and K-FABP inhibit PPAR activity nonselectively (18
) and the data in the present report is not clear to us.
Here, we provide evidence that some FABPs act in concert with PPARs and that this activity is highly selective for particular FABP-PPAR pairs: A-FABP specifically enhances the activity of PPARγ, while K-FABP activates PPARβ. The data demonstrate further that A-FABP and K-FABP relocate from the cytosol to the nucleus in response to particular PPAR ligands and that the nuclear localization of K-FABP is critical for allowing this protein to augment the activity of its cognate receptor. Interestingly, we found that both the ligand-induced nuclear localization of the FABPs and their ability to enhance transcriptional activity are highly selective for the ligand of a particular PPAR isotype. This is so despite the apparent lack of selectivity in ligand binding by the FABPs (Table ). These observations suggest that FABPs employ different modes of binding toward different ligands. In other words, they suggest that some ligands induce correct alterations in the FABP structure, leading to activation of the proteins, while others do not.
In addition to their functional interactions, A-FABP and K-FABP were found to physically associate with PPARγ and PPARβ, respectively. These interactions were specific for particular protein pairs and depended on the presence of particular ligands. The observed FABP-PPAR interactions were found to be weak, suggesting that the resulting complex is transient in nature, i.e., it comprises a short-lived intermediate which dissociates rapidly following transfer of the ligand. Nevertheless, examination of the mechanism by which a ligand moves from FABPs to PPARγ established that direct and selective FABP-PPAR interactions do occur and are functionally important. These studies revealed that A-FABP delivers troglitazone to PPARγ through direct association between the binding protein and the receptor. The selectivity of these interactions was confirmed by the observations that, unlike A-FABP, K-FABP does not interact with PPARγ, i.e., movement of troglitazone from K-FABP to the receptor proceeds through simple diffusion. A-FABP-mediated channeling of ligands to PPARγ significantly facilitated the ligation of the receptor, providing a rationale for understanding the ability of this binding protein (which is not shared by K-FABP) to enhance PPARγ transcriptional activity. A-FABP and K-FABP thus appear to function in a manner similar to that of CRABP-II in regulating the activity of nuclear hormone receptors, i.e., their association with particular ligands in the cytosol leads to translocalization into the nucleus, where they form a short-lived complex with a cognate PPAR. This complex mediates ligand channeling to the receptor, thereby facilitating its activation and enhancing its transcriptional activity. The importance of FABPs for biological activities mediated by PPARs is demonstrated by the observation that the presence of K-FABP is essential for the ability of PPARβ to properly induce differentiation of keratinocytes. Taken together, the data reveal that the A-FABP-PPARγ and K-FABP-PPARβ pairs cooperate tightly in regulating transcription in adipocytes and keratinocytes, respectively. Hence, the tissue-specific functions of PPARs are found to be closely supported by the tissue-specific expression of particular FABPs.
Functional interactions between FABPs and PPARs are also suggested by examination of mice and cells that are deficient in FABP expression. It has recently been reported that PPARβ plays a central role in neuronal differentiation (39
) and that reduction in K-FABP expression in the neuronal cell line PC12 results in inhibition of differentiation of these cells (1
). Taken together with our data demonstrating the importance of K-FABP and its cognate receptor, PPARβ, in inducing differentiation in keratinocytes, these observations raise the intriguing possibility that the same FABP-PPAR pair plays a similar role in mediating neuronal differentiation. Regarding A-FABP, it has been reported that mice in whom expression of this protein has been inhibited, like their wild-type counterparts, develop obesity when fed with a high-fat diet. However, unlike control mice, A-FABP−/−
animals do not become insulin resistant or diabetic. This striking phenotype was observed despite the fact that disruption of A-FABP in the null mice is compensated for by up-regulation of K-FABP in their adipose tissues (20
). Interestingly, mice that are heterozygous for PPARγ, like A-FABP-null mice, display increased insulin sensitivity compared to wild-type animals (32
). Although the exact role of PPARγ in regulating insulin sensitivity is not clear at present, the similarity of the phenotypes of A-FABP−/−
mice and mice deficient in PPARγ expression suggests that the two proteins act in concert. In view of the present study, the observation that up-regulation of K-FABP expression does not functionally compensate for the loss of A-FABP in A-FABP−/−
mice appears to reflect the PPAR selectivity of these FABPs. It should also be noted that PPARγ has been reported to be essential for adipocyte differentiation both in vitro and in vivo (38
), while A-FABP-null mice do not display an adipose tissue deficit (20
). This apparent conflict is resolved by considering that the hypothesis put forward here does not propose that FABPs are absolutely essential for PPAR activity under all conditions. Clearly, PPARs can be activated by ligands in the absence of a binding protein, most likely due to the ability of these small ligands to enter the nucleus by free diffusion. Our data indicate, however, that FABPs function by facilitating ligand delivery, i.e., they act to enhance transcriptional activation by providing increased fluxes. Indeed, we show that the potentiating activity of FABP is especially significant when cellular levels of ligands are limiting, i.e., under conditions that necessitate enhanced efficiency of ligand delivery. These observations suggest that while FABPs are dispensable at high ligand concentrations, they become essential at low concentrations. A prediction that can be derived from these observations is that mice lacking A-FABP will display a more severe phenotype under conditions of ligand deficiency.
While the present study establishes that some FABPs physically and functionally cooperate with specific PPARs, several important questions remain to be explored. For example, the mechanisms through which FABPs locate to the nucleus in response to correct ligands is not understood at present. Similarly, considering the close similarities of the overall folding of FABPs, the specific structural features that allow them to discriminate between particular PPAR isotypes remain to be elucidated. Finally, it is worth noting that the existence of at least nine isotypes of FABP suggest that some of these proteins may have roles that are different from the functions of A-FABP and K-FABP described here. The complete scope of the biological functions of FABPs has thus only begun to be elucidated.