Correction to: Partitioning of adipose lipid metabolism by altered expression and function of PPAR isoforms after bariatric surgery.

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Partitioning of adipose lipid metabolism by altered expression and function of PPAR isoforms after bariatric surgery.

BACKGROUND: Bariatric surgery remains the most effective treatment for reducing adiposity and eliminating type 2 diabetes; however, the mechanism(s) responsible have remained elusive. Peroxisome proliferator-activated receptors (PPAR) encompass a family of nuclear hormone receptors that upon activation exert control of lipid metabolism, glucose regulation and inflammation. Their role in adipose tissue following bariatric surgery remains undefined. MATERIALS AND METHODS: Subcutaneous adipose tissue biopsies and serum were obtained and evaluated from time of surgery and on postoperative day 7 in patients randomized to Roux-en-Y gastric bypass (n=13) or matched caloric restriction (n=14), as well as patients undergoing vertical sleeve gastrectomy (n=33). Fat samples were evaluated for changes in gene expression, protein levels, ß-oxidation, lipolysis and cysteine oxidation. RESULTS: Within 7 days, bariatric surgery acutely drives a change in the activity and expression of PPARγ and PPARδ in subcutaneous adipose tissue thereby attenuating lipid storage, increasing lipolysis and potentiating lipid oxidation. This unique metabolic alteration leads to changes in downstream PPARγ/δ targets including decreased expression of fatty acid binding protein (FABP) 4 and stearoyl-CoA desaturase-1 (SCD1) with increased expression of carnitine palmitoyl transferase 1 (CPT1) and uncoupling protein 2 (UCP2). Increased expression of UCP2 not only facilitated fatty acid oxidation (increased 15-fold following surgery) but also regulated the subcutaneous adipose tissue redoxome by attenuating protein cysteine oxidation and reducing oxidative stress. The expression of UCP1, a mitochondrial protein responsible for the regulation of fatty acid oxidation and thermogenesis in beige and brown fat, was unaltered following surgery. CONCLUSIONS: These results suggest that bariatric surgery initiates a novel metabolic shift in subcutaneous adipose tissue to oxidize fatty acids independently from the beiging process through regulation of PPAR isoforms. Further studies are required to understand the contribution of this shift in expression of PPAR isoforms to weight loss following bariatric surgery.

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.

Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase.

FATP4 (SLC27A4) is a member of the fatty acid transport protein (FATP) family, a group of evolutionarily conserved proteins that are involved in cellular uptake and metabolism of long and very long chain fatty acids. We cloned and characterized the murine FATP4 gene and its cDNA. From database analysis we identified the human FATP4 genomic sequence. The FATP4 gene was assigned to mouse chromosome 2 band B, syntenic to the region 9q34 encompassing the human gene. The open reading frame was determined to be 1929 bp in length, encoding a polypeptide of 643 amino acids. Within the coding region, the exon-intron structures of the murine FATP4 gene and its human counterpart are identical, revealing a high similarity to the FATP1 gene. The overall amino acid identity between the deduced murine and human FATP4 polypeptides is 92.2%, and between the murine FATP1 and FATP4 polypeptides is 60.3%. Northern analysis showed that FATP4 mRNA was expressed most abundantly in small intestine, brain, kidney, liver, skin and heart. Transfection of FATP4 cDNA into COS1 cells resulted in a 2-fold increase in palmitoyl-CoA synthetase (C16:0) and a 5-fold increase in lignoceroyl-CoA synthetase (C24:0) activity from membrane extracts, indicating that the FATP4 gene encodes an acyl-CoA synthetase with substrate specificity biased towards very long chain fatty acids.

Upregulation of bone morphogenetic protein GDF-3/Vgr-2 expression in adipose tissue of FABP4/aP2 null mice.

High-fat-fed C57Bl/6J FABP4/aP2 null mice develop obesity but not the related hyperglycemia or hyperinsulinemia characteristic of type II diabetes. FABP4/aP2 protein's function to bind fatty acids in the adipocytes may promote total body energy homeostasis by linking energy depots to the ability to express signaling molecules similar to leptin. To test this hypothesis, proteomic analysis of serum proteins from high-fat-fed wild-type and FABP4/aP2 null mice revealed that the GDF-3/Vgr-2 protein, a bone morphogenetic protein, was upregulated in C57Bl/6J FABP4/aP2 null mice. The increase in serum GDF-3/Vgr-2 protein was correlated with a 27-fold increase in adipose GDF-3/Vgr-2 mRNA. In contrast, leptin expression was unaltered between FABP4/aP2 null and wild-type animals. The expression of GDF-3/Vgr-2 mRNA was not substantially different in adipose tissue of db/db and tb/tb mice compared to wild-type controls. The expression of GDF-3/Vgr-2 mRNA was dependent upon the age and diet of the animals, declining as a function of age in high-fat-fed wild-type animals while increasing in the FABP4/aP2 null strain. These results identify GDF-3/Vgr-2 as an age- and fat-regulated, adipose-derived cytokine suggesting a linkage between adipocyte fatty acid metabolism and the expression of the bone morphogenetic family of differentiation regulators.

Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm.

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARgamma plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.

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.

The mammalian fatty acid-binding protein multigene family: molecular and genetic insights into function.

Intracellular fatty acid-binding proteins associate with fatty acids and other hydrophobic biomolecules in an internal cavity, providing for solubilization and metabolic trafficking. Analyses of their in vivo function by molecular and genetic techniques reveal specific function(s) that fatty acid-binding proteins perform with respect to fatty acid uptake, oxidation and overall metabolic homeostasis.

The fatty acid transport protein (FATP1) is a very long chain acyl-CoA synthetase.

The primary sequence of the murine fatty acid transport protein (FATP1) is very similar to the multigene family of very long chain (C20-C26) acyl-CoA synthetases. To determine if FATP1 is a long chain acyl coenzyme A synthetase, FATP1-Myc/His fusion protein was expressed in COS1 cells, and its enzymatic activity was analyzed. In addition, mutations were generated in two domains conserved in acyl-CoA synthetases: a 6- amino acid substitution into the putative active site (amino acids 249-254) generating mutant M1 and a 59-amino acid deletion into a conserved C-terminal domain (amino acids 464-523) generating mutant M2. Immunolocalization revealed that the FATP1-Myc/His forms were distributed between the COS1 cell plasma membrane and intracellular membranes. COS1 cells expressing wild type FATP1-Myc/His exhibited a 3-fold increase in the ratio of lignoceroyl-CoA synthetase activity (C24:0) to palmitoyl-CoA synthetase activity (C16:0), characteristic of very long chain acyl-CoA synthetases, whereas both mutant M1 and M2 were catalytically inactive. Detergent-solubilized FATP1-Myc/His was partially purified using nickel-based affinity chromatography and demonstrated a 10-fold increase in very long chain acyl-CoA specific activity (C24:0/C16:0). These results indicate that FATP1 is a very long chain acyl-CoA synthetase and suggest that a potential mechanism for facilitating mammalian fatty acid uptake is via esterification coupled influx.

Fatty acid trafficking in the adipocyte.

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.

Biochemical and biophysical analysis of the intracellular lipid binding proteins of adipocytes.

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.

Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydrophobic ligands. The interaction was demonstrated in vitro by the binding of ALBP to HSL translated in vitro, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue. Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies. The HSL-ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain. These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids.

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.

Identification of a functional peroxisome proliferator-responsive element in the murine fatty acid transport protein gene.

Fatty acid transport protein (FATP), a plasma membrane protein implicated in controlling adipocyte transmembrane fatty acid flux, is up-regulated as a consequence of adipocyte differentiation and down-regulated by insulin. Based upon the sequence of the FATP gene upstream region (Hui, T. Y., Frohnert, B. I., Smith, A. J., Schaffer, J. A., and Bernlohr, D. A. (1998) J. Biol. Chem. 273, 27420-27429) a putative peroxisome proliferator-activated receptor response element (PPRE) is present from -458 to -474. To determine whether the FATP PPRE was functional, and responded to lipid activators, transient transfection of FATP-luciferase reporter constructs into CV-1 and 3T3-L1 cells was carried out. In CV-1 cells, FATP-luciferase activity was up-regulated 4- and 5.5-fold, respectively, by PPARalpha and PPARgamma in the presence of their respective activators in a PPRE-dependent mechanism. PPARdelta, however, was unable to mediate transcriptional activation under any condition. In 3T3-L1 cells, the PPRE conferred a small but significant increase in expression in preadipocytes, as well as a more robust up-regulation of FATP expression in adipocytes. Furthermore, the PPRE conferred the ability for luciferase expression to be up-regulated by activators of both PPARgamma and retinoid X receptor alpha (RXRalpha) in a synergistic manner. PPARalpha and PPARdelta activators did not up-regulate FATP expression in 3T3-L1 adipocytes, however, suggesting that these two subtypes do not play a significant role in differentiation-dependent activation in fat cells. Electromobility shift assays showed that all three PPAR subtypes were able to bind specifically to the PPRE as heterodimers with RXRalpha. Nuclear extracts from 3T3-L1 adipocytes also showed a specific gel-shift complex with the FATP PPRE. To correlate the expression of FATP to its physiological function, treatment of 3T3-L1 adipocytes with PPARgamma and RXRalpha activators resulted in an increased uptake of oleate. Moreover, linoleic acid, a physiological ligand, up-regulated FATP expression 2-fold in a PPRE-dependent manner. These results demonstrate that the FATP gene possesses a functional PPRE and is up-regulated by activators of PPARalpha and PPARgamma, thereby linking the activity of the protein to the expression of its gene. Moreover, these results have implications for the mechanism by which certain PPARgamma activators such as the antidiabetic thiazolidinedione drugs affect adipose lipid metabolism.

Surface properties of adipocyte lipid-binding protein: Response to lipid binding, and comparison with homologous proteins.

Adipocyte lipid-binding protein (ALBP) is one of a family of intracellular lipid-binding proteins (iLBPs) that bind fatty acids, retinoids, and other hydrophobic ligands. The different members of this family exhibit a highly conserved three-dimensional structure; and where structures have been determined both with (holo) and without (apo) bound lipid, observed conformational changes are extremely small (Banaszak, et al., 1994, Adv. Prot. Chem. 45, 89; Bernlohr, et al., 1997, Annu. Rev. Nutr. 17, 277). We have examined the electrostatic, hydrophobic, and water accessible surfaces of ALBP in the apo form and of holo forms with a variety of bound ligands. These calculations reveal a number of previously unrecognized changes between apo and holo ALBP, including: 1) an increase in the overall protein surface area when ligand binds, 2) expansion of the binding cavity when ligand is bound, 3) clustering of individual residue exposure increases in the area surrounding the proposed ligand entry portal, and 4) ligand-binding dependent variation in the topology of the electrostatic potential in the area surrounding the ligand entry portal. These focused analyses of the crystallographic structures thus reveal a number of subtle but consistent conformational and surface changes that might serve as markers for differential targeting of protein-lipid complexes within the cell. Most changes are consistent from ligand to ligand, however there are some ligand-specific changes. Comparable calculations with intestinal fatty-acid-binding protein and other vertebrate iLBPs show differences in the electrostatic topology, hydrophobic topology, and in localized changes in solvent exposure near the ligand entry portal. These results provide a basis toward understanding the functional and mechanistic differences among these highly structurally homologous proteins. Further, they suggest that iLBPs from different tissues exhibit one of two predominant end-state structural distributions of the ligand entry portal.

Regulation of adipocyte gene expression by polyunsaturated fatty acids.

A wide number of adipocyte genes are regulated by exogenous polyunsaturated fatty acids (PUFA) through the actions of the peroxisome proliferator activated receptor. Such genes include the adipocyte lipid-binding protein (ALBP or aP2) which plays a central role in facilitating the trafficking of fatty acids within adipocytes. Work from a number of laboratories has suggested the key elements of the lipid signal transduction pathway include: (1) the transport of exogenous PUFAs across the plasma membrane, (2) metabolism of polyunsaturated fatty acids to second messengers including 15-deoxy delta 12,14 prostaglandin J2 (15dPGJ2), (3) trafficking of 15dPGJ2 and other second messengers from the smooth ER to the nucleus for association with peroxisome proliferator activated receptor gamma (PPAR gamma), and (4) dimerization of PPAR gamma with retinoid X receptor (RXR) permitting regulation of transcription via association with any of several nuclear co-activators or repressors. In addition to the aP2 gene being a target of activation by fatty acids, at the protein level ALBP/aP2 plays a role in trafficking of fatty acids and/or their metabolises. We report here that in a heterologous system using CV-1 cells transiently transfected with PPAR gamma 2, co-expression of ALBP/aP2 enhances the PPAR-dependent activation of gene transcription. These results suggest that ALBP/aP2 functions as a positive factor in fatty acid signalling by directly targetting and delivering fatty acids metabolites to the lipid signal transduction pathway.

Cloning and chromosomal location of the murine keratinocyte lipid-binding protein gene.

The keratinocyte lipid-binding protein (KLBP) is a member of a large multigene family of intracellular fatty-acid-binding proteins. It is expressed in skin and tongue epithelia, adipose, lung and mammary tissue and has been found upregulated in several skin cell carcinomas and papillomas (Krieg et al., 1993). In order to study the regulation of KLBP expression, the murine gene has been cloned. Southern analysis using an exon 2 specific cDNA probe indicated the presence of multiple copies of the gene in the murine genome. Based on the highly conserved structure of the fatty-acid-binding protein genes, the third intron of the KLBP gene was PCR-amplified utilizing murine genomic DNA. Southern analysis with the intron 3 probe identified one unique gene in the murine genome. A full-length genomic clone of KLBP was obtained from a P1 library, and the structural gene was sequenced. Similar to the other FABP genes, the functional KLBP gene contains four exons separated by three introns and maintains the conservation of size and placement of each exon. A functional minimal promoter was demonstrated by transient transfections of 5' upstream KLBP-luciferase reporter constructs into line 308 keratinocyte cells as well as in primary adipocytes. RT-PCR on primary adipocyte RNA demonstrated expression of this KLBP gene by amplification of intron 3 from the primary transcript. Fluorescence in-situ hybridization identified the murine KLBP gene as the fourth FABP gene on chromosome 3, along with myelin P2, ALBP, and intestinal FABP. These studies provide a framework for analysis of KLBP expression in normal and pathophysiological conditions.

Characterization of the murine fatty acid transport protein gene and its insulin response sequence.

Fatty acid transport protein (FATP) was identified by expression cloning strategies (Schaffer, J. E., and Lodish, H. F. (1994) Cell 79, 427-436) and shown by transfection analysis to catalyze the transfer of long-chain fatty acids across the plasma membrane of cells. It is expressed highly in tissues exhibiting rapid fatty acid metabolism such as skeletal muscle, heart, and adipose. FATP mRNA levels are down-regulated by insulin in cultured 3T3-L1 adipocytes and up-regulated by nutrient depletion in murine adipose tissue (Man, M. Z., Hui, T. Y., Schaffer, J. E., Lodish, H. F., and Bernlohr, D. A. (1996) Mol. Endocrinol. 10, 1021-1028). To determine the molecular mechanism of insulin regulation of FATP transcription, we have isolated the murine FATP gene and its 5'-flanking sequences. The FATP gene spans approximately 16 kilobases and contains 13 exons, of which exon 2 is alternatively spliced. S1 nuclease and RNase protection assays revealed the presence of multiple transcription start sites; the DNA sequence upstream of the predominant transcription start sites lacks a typical TATA box. By transient transfection assays in 3T3-L1 adipocytes, the inhibitory action of insulin on FATP transcription was localized to a cis-acting element with the sequence 5'-TGTTTTC-3' from -1347 to -1353. This sequence is very similar to the insulin response sequence found in the regulatory region of other genes negatively regulated by insulin such as those encoding phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, and insulin-like growth factor-binding protein 1. Fluorescence in situ hybridization analysis revealed that the murine FATP gene is localized to chromosome 8, band 8B3.3. Interestingly, this region of chromosome 8 contains a cluster of three other genes important for fatty acid homeostasis, lipoprotein lipase, the mitochondrial uncoupling protein 1 (UCP1) and sterol regulatory element-binding protein 1. These results characterize the murine FATP gene and its insulin responsiveness as well as present a framework for future studies of its role in lipid metabolism, obesity, and type II diabetes mellitus.

Analysis of a series of phenylalanine 57 mutants of the adipocyte lipid-binding protein.

The importance of phenylalanine 57, an adipocyte lipid-binding protein (ALBP) portal residue, to ligand affinity and specificity has been investigated using a series of ALBP position 57 mutants. In wild-type ALBP, phenylalanine 57 undergoes a side chain rotation upon ligand binding, moving from an inwardly oriented, ligand-exclusive position in apoprotein structures to an outwardly oriented position in the holoprotein. To examine the role of F57 side chain rotation in the apoprotein-holoprotein transition and in ligand selectivity, ALBP site-specific mutants F57A, F57G, F57H, and F57W were expressed in Escherichia coli and purified to homogeneity. Mutants were analyzed for binding characteristics and stability toward chemical denaturation, and energy-minimized models of each mutant were constructed using apo, oleate-, and arachidonate-bound ALBP crystallographic coordinates. The stability of ALBP forms (wtALBP approximately F57G > F57A > F57W > F57H) was unrelated to the affinity of ALBP forms (wtALBP approximately F57W > F57H > F57G > F57A) for various lipids and did not vary between fatty acids. Since ligand selectivity was maintained between wild type and all mutants while ligand affinity was grossly diminished, we conclude that phenylalanine 57 is critical to the formation of the fatty acid/ALBP complex, but is uninvolved in determination of selectivity over the range of physiological ligands tested.