Heme-binding by Drosophila retinoid- and fatty acid-binding glycoprotein (RFABG), a member of the proapolipophorin gene family.
(17/1344)
We previously have cloned and characterized a retinoid- and fatty acid-binding glycoprotein (RFABG) isolated from the heads of Drosophila melanogaster. The protein is composed of two glycosylated subunits (Mr = >200,000 and 70,000) and is a member of the proapolipophorin gene family. Spectral analysis of purified RFABG revealed an absolute absorbance peak at 405 nm, which is typical for a heme-containing protein. The aim of the present study was to characterize the heme-binding properties of RFABG. Upon saturation of the protein solution with carbon monoxide followed by dithionite reduction, a red shift of the Soret peak to 424 nm and the characteristic alpha- and beta- bands at 567 and 539 nm were observed. Native RFABG contains approximately 0.175 moles of heme (mol/mol) indicating that purified RFABG is primarily the apoprotein. Hemin-agarose affinity chromatography of the native RFABG followed by Western blot analysis showed a single immunoreactive band at 70 kDa, indicating that the heme-binding domain resides in the 70 kDa subunit. Although retinoid and fatty acid also bind to the 70 kDa subunit, no competition was observed when an excess of heme was added to a solution of retinoid or fatty acid bound to RFABG. Heme added to a solution of purified RFABG bound in a saturable manner with an affinity of 3.8 x 10(-7) m.Thus, the current study clearly demonstrates that retinoid- and fatty acid-binding glycoprotein is a novel heme-binding protein, which may be involved in the transport and/or metabolism of heme in Drosophila. (+info)
Training-induced elevation in FABP(PM) is associated with increased palmitate use in contracting muscle.
(18/1344)
To evaluate the effects of endurance training in rats on fatty acid metabolism, we measured the uptake and oxidation of palmitate in isolated rat hindquarters as well as the content of fatty acid-binding proteins in the plasma membranes (FABP(PM)) of red and white muscles from 16 trained (T) and 18 untrained (UT) rats. Hindquarters were perfused with 6 mM glucose, 1,800 microM palmitate, and [1-(14)C]palmitate at rest and during electrical stimulation (ES) for 25 min. FABP(PM) content was 43-226% higher in red than in white muscles and was increased by 55% in red muscles after training. A positive correlation was found to exist between succinate dehydrogenase activity and FABP(PM) content in muscle. Palmitate uptake increased by 64-73% from rest to ES in both T and UT and was 48-57% higher in T than UT both at rest (39.8 +/- 3.5 vs. 26.9 +/- 4. 4 nmol. min(-1). g(-1), T and UT, respectively) and during ES (69.0 +/- 6.1 vs. 43.9 +/- 4.4 nmol. min(-1). g(-1), T and UT, respectively). While the rats were resting, palmitate oxidation was not affected by training; palmitate oxidation during ES was higher in T than UT rats (14.8 +/- 1.3 vs. 9.3 +/- 1.9 nmol. min(-1). g(-1), T and UT, respectively). In conclusion, endurance training increases 1) plasma free fatty acid (FFA) uptake in resting and contracting perfused muscle, 2) plasma FFA oxidation in contracting perfused muscle, and 3) FABP(PM) content in red muscles. These results suggest that an increased number of these putative plasma membrane fatty acid transporters may be available in the trained muscle and may be implicated in the regulation of plasma FFA metabolism in skeletal muscle. (+info)
Expression of peroxisome proliferator-activated receptor PPARdelta promotes induction of PPARgamma and adipocyte differentiation in 3T3C2 fibroblasts.
(19/1344)
Nutritional long chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. The molecular mechanisms implicated in this action of fatty acids remain poorly understood. It has been well established that peroxisome proliferator-activated receptor (PPAR) gamma, activated by specific prostanoids, plays a central role in the control of adipocyte gene expression and terminal differentiation. Thus far, the role of PPARdelta in the control of adipose tissue mass has remained unclear. Herein, we report the effects of ectopically expressed PPARdelta on the control of adipose-related gene expression and adipogenesis of 3T3C2 fibroblasts. Treatment of PPARdelta-expressing fibroblasts with fatty acids alone did not stimulate adipogenesis, whereas exposure of cells to a combination of fatty acids and PPARgamma activators promoted lipid accumulation and expression of a typical adipocyte program. At the molecular level, activation of PPARdelta by fatty acids induced transcription of the genes encoding fatty acid transporter, adipocyte lipid-binding protein, and PPARgamma. Subsequent activation of PPARgamma by specific agonists appeared to be required to promote terminal differentiation. These data demonstrate that PPARgamma gene expression is under the control of PPARdelta activated by fatty acids and could explain, at least partially, the adipogenic action of nutritional fatty acids. (+info)
Studies of the ligand binding reaction of adipocyte lipid binding protein using the fluorescent probe 1, 8-anilinonaphthalene-8-sulfonate.
(20/1344)
The fluorescent probe anilinonaphthalene-8-sulfonate binds to adipocyte lipid binding protein at a site that competes with normal physiological ligands, such as fatty acids. Binding to the protein is accompanied by a relatively large increase in fluorescent intensity. To correlate the major change in optical properties and to determine the mechanism of competitive inhibition with fatty acids, the crystal structure of the protein with the bound fluorophore has been determined. In addition, the thermodynamic contributions to the binding reaction have been studied by titration calorimetry. Because the binding site is in a relatively internal position, kinetic studies have also been carried out to determine k(on). The results indicate that binding is not accompanied by any major conformational change. However, the negatively charged sulfonate moiety is not positioned the same as the carboxylate of fatty acid ligands as determined in previous studies. Nonetheless, the binding reaction is still driven by enthalpic effects. As judged by the crystallographic structure, a significant amount of the surface of the fluorophore is no longer exposed to water in the bound state. (+info)
Cellular uptake and intracellular trafficking of long chain fatty acids.
(21/1344)
While aspects of cellular fatty acid uptake have been studied as early as 50 years ago, recent developments in this rapidly evolving field have yielded new functional insights on the individual mechanistic steps in this process. The extremely low aqueous solubility of long chain fatty acids (LCFA) together with the very high affinity of serum albumin and cytoplasmic fatty acid binding proteins for LCFA have challenged the limits of technology in resolving the individual steps of this process. To date no single mechanism alone accounts for regulation of cellular LCFA uptake. Key regulatory points in cellular uptake of LCFA include: the aqueous solubility of the LCFA; the driving force(s) for LCFA entry into the cell membrane; the relative roles of diffusional and protein mediated LCFA translocation across the plasma membrane; cytoplasmic LCFA binding protein-mediated uptake and/or intracellular diffusion; the activity of LCFA-CoA synthetase; and cytoplasmic protein mediated targeting of LCFA or LCFA-CoAs toward specific metabolic pathways. The emerging picture is that the cell has multiple, overlapping mechanisms that assure adequate uptake and directed intracellular movement of LCFA required for maintenance of physiological functions. The upcoming challenge is to take advantage of new advances in this field to elucidate the differential interactions between these pathways in intact cells and in tissues. (+info)
Conjugated linoleic acid is a potent naturally occurring ligand and activator of PPARalpha.
(22/1344)
We have previously shown that a mixture of dietary conjugated derivatives of linoleic acid (conjugated linoleic acid, CLA) induces peroxisome proliferator-responsive enzymes and modulates hepatic lipid metabolism in vivo. The present studies demonstrate that CLA is a high affinity ligand and activator of peroxisome proliferator-activated receptor alpha (PPARalpha) and induces accumulation of PPAR-responsive mRNAs in a rat hepatoma cell line. Using a scintillation proximity assay (SPA), CLA isomers were shown to be ligands for human PPARalpha with a rank order of potency of (9Z,11E)>(10E,12Z)>(9E,11E)> furan-CLA (IC(50) values from 140 nm to 400 nm). Levels of acyl-CoA oxidase (ACO), liver fatty acid-binding protein (L-FABP), and cytochrome P450IVA1 (CYP4A1) mRNA were induced by CLA in FaO hepatoma cells. Even though linoleate and CLA were incorporated into lipids of hepatoma cells to the same extent, linoleate had little or no effect on ACO, CYP4A1, or L-FABP mRNA. In agreement with its binding potency, (9Z,11E)-CLA was the most efficacious PPARalpha activator in the mouse PPARalpha-GAL4(UAS)(5)-CAT reporter system. These data indicate that CLA is a ligand and activator of PPARalpha and its effects on lipid metabolism may be attributed to transcriptional events associated with this nuclear receptor. Also, (9Z,11E)-CLA is one of the most avid fatty acids yet described as a PPARalpha ligand. (+info)
Type 2 diabetes mellitus: association study of five candidate genes in an Indian population of Guadeloupe, genetic contribution of FABP2 polymorphism.
(23/1344)
We studied by PCR-RFLP 6 polymorphisms in these 5 candidate genes: Ala54Thr in the fatty acid binding protein 2 gene (FABP2), A to G substitution in the uncoupling protein type 1 gene (UCP1), Asp905Tyr in the protein phosphatase type 1 gene (PP1G), Trp64Arg in the human beta 3 adrenergic receptor gene (beta 3AR) and 2 RFLP sites of the vitamin D receptor (VDR) gene (VDRTaq1 and VDRApa1). This study was conducted among 89 cases and 100 controls matched according to age, gender and absence of first degree family link (11 triplets with 2 controls for 1 case and 78 pairs with 1 control for 1 case). Cases and controls were taken among a sample of 429 individuals selected for the study of the prevalence of diabetes in this ethnic group from Guadeloupe. By conditional logistic regression analysis, there was a significant relation (p = 0.02) between the Ala54Thr FABP2 polymorphism and Type 2 DM. Multivariate analysis discriminate the FABP2 polymorphism (p = 0.10), a triglyceridemia over 2 g/l (p < 10(-3)) and high blood pressure (p = 10(-2)) as variables associated with Type 2 DM in this population. These findings suggest that FABP2 does not represent a major gene for Type 2 DM in this migrant Indian population living in Guadeloupe, but seems to be related to the metabolic insulin resistance syndrome. (+info)
Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein.
(24/1344)
The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP. (+info)