Role of adipocyte lipid-binding protein (ALBP) and acyl-CoA binding protein (ACBP) in PPAR-mediated transactivation

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that are activated by a number of fatty acids and fatty acid derivatives. By contrast, we have recently shown that acyl-CoA esters display PPAR antagonistic properties in vitro. We have also shown that the adipocyte lipid binding protein (ALBP), the keratinocyte lipid binding protein (KLBP) and the acyl-CoA binding protein (ACBP) exhibit a prominent nuclear localization in differentiating 3T3-L1 adipocytes. Similarly, ectopic expression of these proteins in CV-1 cells resulted in a primarily nuclear localization. We therefore speculated that FABPs and ACBP might regulate the availability of PPAR agonists and antagonists by affecting not only their esterification in the cytoplasm but also their transport to and availability in the nucleus. We show here that coexpression of ALBP or ACBP exerts a negative effect on ligand-dependent PPAR transactivation, when tetradecylthioacetic (TTA) is used as ligand but not when the thiazolidinedione BRL49653 is used as ligand. The results presented here do not support the hypothesis that ALBP facilitates the transport of the fatty acid-type ligands to the nucleus, rather ALBP appears to sequester or increase the turn-over of the agonist. Similarly, our results are in keeping with a model in which ACBP increase the metabolism of these ligands.     

Reduced activity of enzymes coupling ATP-generating with ATP-consuming processes in the failing myocardium

Adipocytes express two lipid-binding proteins; the major one termed the adipocyte lipid-binding protein or aP2 (ALBP/aP2) and a minor one referred to as the keratinocyte lipid-binding protein (KLBP). In order to evaluate the potential physiological roles for these proteins, their biochemical and biophysical properties have been analyzed and compared. ALBP/aP2 and KLBP exhibit similar binding affinities for most long-chain fatty acids; however, ALBP/aP2 exhibits a two to three-fold increased affinity for myristic, palmitic, oleic and linoleic acids, the predominant fatty acids of adipocytes. As measured by guanidinium hydrochloride denaturation, the stability of ALBP/aP2 is nearly 3 kcal/mol greater than that of KLBP. While the pI of ALBP/aP2 was determined to be 9.0, that of KLBP is 6.5 suggesting differing net charges at physiological pH. Analysis of surface electrostatic properties of ALBP/aP2 and KLBP revealed similar charge polarity, although differences in the detailed charge distribution exist between the proteins. The distribution of hydrophobic patches was also different between the proteins, ALBP/aP2 has only scattered hydrophobic surfaces while KLBP has a large hydrophobic patch near the ligand portal into the binding cavity. In sum, these results point out that despite the striking similarity between ALBP/aP2 and KLBP in tertiary structure, significant differences in ligand binding and surface properties exist between the two proteins. Hence, while it is tempting to speculate that ALBP/aP2 and KLBP are metabolically interchangeable, careful analysis suggests that the two proteins are quite distinct and likely to play unique metabolic roles.     

Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels

The availability of mice containing an adipocyte lipid-binding protein (ALBP/aP2) gene disruption allowed for a direct examination of the presumed role of lipid-binding proteins in the mobilization and trafficking of intracellular fatty acids. Total body and epididymal fat pad weights, as well as adipose cell morphology, were unaltered in male ALBP/aP2 disrupted mice when compared to their wild-type littermates. Analysis of adipocytes isolated from wild-type and ALBP/aP2 null mice revealed that a selective 40- and 13-fold increase in the level of the keratinocyte lipid-binding protein (KLBP) mRNA and protein, respectively, accompanied the ALBP/aP2 gene disruption. Although KLBP protein was significantly up-regulated, the total lipid-binding protein level decreased 8 -fold as a consequence of the disruption. There was no appreciable difference in the rate of fatty acid influx or esterification in adipocytes of wild-type and ALBP/aP2 null animals. To the contrary, basal lipolysis decreased approximately 40% in ALBP/aP2 nulls as compared to wild-type littermates. The glycerol release from isproterenol-stimulated ALBP/aP2 null fat cells was similarly reduced by approximately 35%. Consistent with a decrease in basal efflux, the non-esterified fatty acid (NEFA) level was nearly 3-fold greater in adipocytes from ALBP/aP2 nulls as compared to wild-type animals. The significant decrease in both basal and isoproterenol-stimulated lipolysis in adipose tissue of ALBP/aP2 null mice supports the model whereby intracellular lipid-binding proteins function as lipid chaperones, facilitating the movement of fatty acids out of the fat cell.     

Characterization of the functional interaction of adipocyte lipid-binding protein with hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is an intracellular lipase that plays an important role in the hydrolysis of triacylglycerol in adipose tissue. HSL has been shown to interact with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that bind fatty acids and other hydrophobic ligands. The current studies have addressed the functional significance of the association and mapped the site of interaction between HSL and ALBP. Incubation of homogeneous ALBP with purified, recombinant HSL in vitro resulted in a 2-fold increase in substrate hydrolysis. Moreover, the ability of oleate to inhibit HSL hydrolytic activity was attenuated by co-incubation with ALBP. Co-transfection of Chinese hamster ovary cells with HSL and ALBP resulted in greater hydrolytic activity than transfection of cells with HSL and vector alone. Deletional mutations of HSL localized the region of HSL that interacts with ALBP to amino acids 192-200, and site-directed mutagenesis of individual amino acids in this region identified His-194 and Glu-199 as critical for mediating the interaction of HSL with ALBP. Interestingly, HSL mutants H194L and E199A, each of which retained normal basal hydrolytic activity, failed to display an increase in hydrolytic activity when co-transfected with wild type ALBP. Therefore, ALBP increases the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and via specific protein-protein interaction. Thus, HSL and ALBP constitute a functionally important lipolytic complex.     

Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages

The adipocyte lipid-binding protein (ALBP/aP2) belongs to a multigene family of fatty acid and retinoid transport proteins. This protein is abundantly expressed in the cytoplasm and nuclear region of adipocytes and is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system for utilization. This report demonstrates that human cholesterol-loaded THP-1 macrophages express ALBP/aP2 and that its expression can be stimulated by oxidized low density lipoprotein (oxLDL). The increase in mRNA expression was paralleled by a similar increase in ALBP/aP2 protein. The increase in ALBP/aP2 mRNA and protein in oxLDL-stimulated THP-1 macrophages is concentration and time dependent and is inhibited by treatment of the cells with an antioxidant inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate (PDTC), and with protein kinase C (PKC) inhibitors bisindolylmaleimide I and Ro-31-8220.These results suggest that activation of both NF-kappaB and PKC signaling pathways is necessary for oxLDL-induced ALBP/aP2 gene expression in THP-1 macrophages and that the upregulation of the fatty acid carrier may be a necessary event in foam cell formation.     

Fatty acid binding protein expression in different human adipose tissue depots in relation to rates of lipolysis and insulin concentration in obese individuals

Two fatty acid binding proteins (FABPs) are expressed in adipose tissue, adipocyte lipid binding protein (ALBP) and keratinocyte lipid binding protein (KLBP). This study investigated FABP expression in visceral and subcutaneous human adipose tissue depots and associations with lipolytic differences between the depots and circulating insulin concentrations. ALBP and KLBP (protein and RNA) were quantified in subcutaneous and omental adipose tissue from obese individuals and expressed relative to actin. ALBP RNA and protein expression was significantly higher in subcutaneous compared to omental adipose tissue (both p < 0.05), whereas KLBP RNA and protein expression was no different between the two sites. There were significant inverse correlations between serum insulin concentrations and the ALBP/KLBP RNA ratio in both subcutaneous and omental adipose tissue (both p < 0.02). Basal rates of glycerol and fatty acid release measured in adipocytes isolated from subcutaneous and omental adipose tissue were significantly higher in the former (p 0.02). Therefore the relative ALBP/KLBP content of human adipose tissue is different in different adipose tissue depots and at the RNA level is related to the circulating insulin concentration, at least in obese subjects. The higher rates of basal lipolysis in adipocytes isolated from subcutaneous compared to omental adipose tissue might be related to the increased ALBP content of the former. Therefore adipose tissue FABPs are interesting candidates for investigation to further our understanding of the insulin resistance syndrome and regulation of lipolysis.     

In vitro phosphorylation of the adipocyte lipid-binding protein (p15) by the insulin receptor. Effects of fatty acid on receptor kinase and substrate phosphorylation.

Phosphorylation of the adipocyte lipid-binding protein (ALBP) isolated from 3T3-L1 cells has been studied in vitro utilizing the wheat germ agglutinin-purified 3T3-L1 adipocyte insulin receptor and the soluble kinase domain of the human insulin receptor. Following insulin-stimulated, ATP-dependent autophosphorylation of the wheat germ agglutinin-purified receptor beta-subunit, ALBP was phosphorylated exclusively on tyrosine 19 in the sequence Glu-Asn-Phe-Asp-Asp-Tyr19, analogous to the substrate phosphorylation consensus sequence observed for several tyrosyl kinases. The concentration of insulin necessary for half-maximal receptor autophosphorylation (KIR0.5) was identical to that necessary for half-maximal ALBP phosphorylation (KALBP0.5), 10 nM. Kinetic analysis indicated that stimulation of ALBP phosphorylation by insulin was attributable to a 5-fold increase in the Vmax (to 0.33 fmol/min/fmol insulin-binding sites) while the Km for ALBP was largely unaffected. By utilizing the soluble kinase domain of the human receptor beta-subunit, the presence of oleate bound to ALBP increased the kcat/Km greater than 3-fold. Oleate dramatically inhibited autophosphorylation of the 38-kDa fragment of the soluble receptor kinase in a concentration dependent fashion (I0.5 approximately 4 microM). The 48-kDa kinase exhibited much less sensitivity to the effects of oleate (I0.5 approximately 190 microM). The inhibition of autophosphorylation of the 48-kDa soluble kinase by oleate was reversed by adding saturating levels of ALBP. These results demonstrate that in vitro the murine adipocyte lipid-binding protein is phosphorylated on tyrosine 19 in an insulin-stimulated fashion by the insulin receptor and that the presence of a bound fatty acid on ALBP increases the affinity of insulin receptor for ALBP. Inhibition of insulin receptor kinase activity by unbound fatty acids suggests that the end products of the lipogenic pathway may feedback inhibit the tyrosyl kinase and that fatty acid-binding proteins have the potential to modulate such interaction.     

Crystal structure of recombinant murine adipocyte lipid-binding protein.

Adipocyte lipid-binding protein (ALBP) is the adipocyte member of an intracellular hydrophobic ligand-binding protein family. ALBP is phosphorylated by the insulin receptor kinase upon insulin stimulation. The crystal structure of recombinant murine ALBP has been determined and refined to 2.5 A. The final R factor for the model is 0.18 with good canonical properties. Crystalline ALBP has a conformation which is essentially identical to that of intestinal fatty acid binding protein and myelin P2 protein. Although the crystal structure is of the apo- form, a cavity resembling that in other family members is present. It contains a number of bound and implied unbound water molecules and shows no large obvious portal to the external milieu. The cavity of ALBP, which by homology is the ligand-binding site, is formed by both polar and hydrophobic residues among which is tyrosine 19. Y19 is phosphorylated by the insulin receptor kinase as described in the accompanying paper [Buelt, M. K., Xu, Z., Banaszak, L. J., & Bernlohr, D. A. (1992) Biochemistry (following paper in this issue)]. By comparing ALBP with the earlier structural results on intestinal fatty acid binding protein, it is now possible to delineate conserved amino acids which help form the binding site in this family.     

Adenovirus-mediated gene transfer in primary murine adipocytes

The transfer of genes into primary murine adipocytes using an adenovirus system has been developed. A recombinant adenovirus was constructed (expressing green fluorescent protein [GFP] under the control of the strong cytomegalovirus [CMV] promoter and a luciferase reporter gene under the control of the weak adipocyte promoter keratinocyte lipid-binding protein [KLBP/FABP5]) and incubated with primary adipocytes from C57BL/6J mice. Analysis of infected cells by confocal microscopy detected GFP expression in both the cytoplasm and nucleus of adipocytes with a 64% efficiency of infection. To demonstrate the applicability of this method in the study of gene regulation, adenovirus-infected adipocytes exhibited significant levels of luciferase activity even from a weak promoter. TPA treatment of infected adipocytes increased luciferase activity, consistent with previous studies indicating that the KLBP/FABP5 gene is up-regulated by phorbol esters.These results provide an efficient, convenient, and sensitive method to transiently infect primary murine adipocytes, facilitating protein expression or permitting analysis of reporter gene activity from both viral and endogenous promoters.     

Structural properties of the adipocyte lipid binding protein

The adipocyte lipid binding protein, ALBP (also adipocyte fatty acid binding protein, A-FABP, 422 protein, aP2, and p15 protein), is one of the most studied of the intracellular lipid binding protein family. Here we sequentially compare the different sources of ALBP and describe the idea that one-third of the amino acid side chains near the N-terminal end appear to play a major role in conformational dynamics and in ligand transfer. Crystallographic data for mouse ALBP are summarized and the ligand binding cavity analyzed in terms of the overall surface and conformational dynamics. The region of the proposed ligand portal is described. Amino acid side chains critical to cavity formation and fatty acid interactions are analyzed by comparing known crystal structures containing a series of different hydrophobic ligands. Finally, we address ALBP ligand binding affinity and thermodynamic studies.     

Chemical phosphorylation of proteins by zinc-ATP

During an examination of in vitro phosphorylation of the adipocyte lipid-binding protein (ALBP) by the insulin receptor, we detected insulin receptor-independent, chemical phosphorylation of ALBP. This activity was present in ALBP purified to homogeneity from murine 3T3-L1 cells and in recombinant murine ALBP purified from expressing E. coli cultures. Phosphoamino acid analysis revealed that chemical phosphorylation of ALBP occurred primarily on Ser residues. The phosphorylation activity occurred in the alkaline pH range from 8 to 11 and exhibited a broad temperature dependence. The reaction rate was linearly dependent upon the ATP concentration and exhibited a biphasic kinetic profile. Eight of twelve other proteins tested also underwent chemical phosphorylation. Zn+2, Mg+2, or Mn+2 promoted optimal phosphorylation of different proteins. We conclude that many proteins are capable of undergoing chemical phosphorylation.     

Purification of murine adipocyte lipid-binding protein. Characterization as a fatty acid- and retinoic acid-binding protein

An adipose-specific protein has been purified from murine 3T3-L1 adipocytes to greater than 98% homogeneity. A purification procedure was developed utilizing a combination of gel filtration, cation exchange chromatography, and covalent chromatography on activated-thiol Sepharose 4B. The protein exists as a single polypeptide with a molecular weight of about 15,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein contains 2 mol of reduced sulfhydryl groups per mol of protein and an amino terminus blocked to sequencing. Automated Edman degradation of trypsin and CNBr-derived peptides has verified that the purified protein is that predicted by the mRNA (Bernlohr, D. A., Angus, C. W., Lane, M. D., Bolanowski, M. A., and Kelly, T. J. Jr. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472). Based on sequence analysis, the 15-kDa adipocyte protein is considered to be a member of a family of tissue-specific, cytosolic lipid-binding proteins. Utilizing a liposome assay, the purified protein binds both oleic acid and retinoic acid saturably with approximately 1 mol of ligand bound per mol of protein. Dissociation constants determined from Scatchard analysis were 3 and 50 microM, respectively. This report represents the first demonstration of a member of this family of structurally related proteins that is capable of binding both fatty acid and retinoic acid. Hence, we propose the name adipocyte lipid-binding protein, or ALBP.     

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.     

The gene encoding acyl-CoA-binding protein is subject to metabolic regulation by both sterol regulatory element-binding protein and peroxisome proliferator-activated receptor alpha in hepatocytes

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular lipid-binding protein that transports acylCoA esters. The protein is expressed in most cell types at low levels; however, expression is particularly high in cells with a high turnover of fatty acids. Here we confirm a previous observation that ACBP expression in rodent liver is down-regulated by fasting, and we show that insulin but not glucose is the inducer of ACBP expression in primary rat hepatocytes. In keeping with the regulation by insulin, we show that ACBP is a sterol regulatory element-binding protein 1c (SREBP-1c) target gene in hepatocytes. Members of the SREBP family activate the rat ACBP gene through binding sites for SREBP and the auxiliary factors Sp1 and nuclear factor Y in the proximal promoter. In addition, we show that ACBP is a peroxisome proliferator-activated receptor (PPAR) alpha target gene in cultured hepatocytes and is induced in the liver by fibrates in a PPARalpha-dependent manner. Thus, ACBP is a dual PPARalpha and SREBP-1c target gene in hepatocytes. Fasting leads to reduced activity of SREBP but increased activity of PPARalpha in hepatocytes, and in keeping with ACBP being a dual target gene, we show that ACBP expression is significantly lower in livers from PPARalpha knock-out mice than in livers from wild type mice. In conclusion, expression of ACBP in rodent hepatocytes is subject to dual metabolic regulation by PPARalpha and SREBP-1c, which may reflect the need for ACBP during lipogenic as well as lipo-oxidative conditions.     

Photoacoustic analysis of proteins: volumetric signals and fluorescence quantum yields.

A series of proteins has been examined using time-resolved, pulsed-laser volumetric photoacoustic spectroscopy. Photoacoustic waveforms were collected to measure heat release for calculation of fluorescence quantum yields, and to explore the possibility of photoinduced nonthermal volume changes occurring in these protein samples. The proteins studied were the green fluorescent protein (GFP); intestinal fatty acid binding protein (IFABP), and adipocyte lipid-binding protein (ALBP), each labeled noncovalently with 1-anilinonaphthalene-8-sulfonate (1,8-ANS) and covalently with 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan); and acrylodan-labeled IFABP and ALBP with added oleic acid. Of this group of proteins, only the ALBP labeled with 1,8-ANS showed significant nonthermal volume changes at the beta = 0 temperature (approximately 3.8 degrees C) for the buffer used (10 mM Tris-HCI, pH 7.5) (beta is the thermal cubic volumetric expansion coefficient). For all of the proteins except for acrylodan-labeled IFABP, the fluorescence quantum yields calculated assuming simple energy conservation were anomalously high, i.e., the apparent heat signals were lower than those predicted from independent fluorescence measurements. The consistent anomalies suggest that the low photoacoustic signals may be characteristic of fluorophores buried in proteins, and that photoacoustic signals derive in part from the microenvironment of the absorbing chromophore.     

Interaction of fatty acids,acyl-CoA derivatives and retinoids with microsomal membranes: effect of cytosolic proteins

This paper reviews characteristics of microsomal membrane structure; long chain fatty acids, acyl CoA derivatives, retinoids and the microsomal formation of acyl CoA derivatives and retinyl esters. It is analyzed how the movement of these molecules at the intracellular level is affected by their respective binding proteins (Fatty acid binding protein, acyl CoA binding protein and cellular retinol binding protein). Studies with model systems using these hydrophobic ligands and the lipid-binding or transfer proteins are also described. This topic is of interest especially because in the esterification of retinol the three substrates and the three binding proteins may interact. (Mol Cell Biochem20: 89–94, 1993)Abbreviations FABP(s) Fatty Acid Binding Protein(s) - CRBP Cellular Retinol Binding Protein - ACBP Acyl-CoA-Binding Protein     

The gene encoding the Acyl-CoA-binding protein is activated by peroxisome proliferator-activated receptor gamma through an intronic response element functionally conserved between humans and rodents

The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals. In vitro studies indicate that ACBP may regulate the availability of acyl-CoA esters for various metabolic and regulatory purposes. The protein is particularly abundant in cells with a high level of lipogenesis and de novo fatty acid synthesis and is significantly induced during adipocyte differentiation. However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown. Here we report that ACBP is a novel peroxisome proliferator-activated receptor (PPAR)gamma target gene. The rat ACBP gene is directly activated by PPARgamma/retinoid X receptor alpha (RXRalpha) and PPARalpha/RXRalpha, but not by PPARdelta/RXRalpha, through a PPAR-response element in intron 1, which is functionally conserved in the human ACBP gene. The intronic PPAR-response element (PPRE) mediates induction by endogenous PPARgamma in murine adipocytes and confers responsiveness to the PPARgamma-selective ligand BRL49653. Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPARgamma/RXR in its natural chromatin context in adipocytes. Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPARgamma-response element.