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• W2050337777 abstract "Fatty acid binding proteins (FAB2222251) are low-molecular-mass, soluble, intracellular lipid carriers. Previous studies on adipocytes from adipocyte fatty acid binding protein (A-FABP)-deficient mice have revealed that both basal and isoproterenol-stimulated lipolysis were markedly reduced (Coe et al. 1999. J. Lipid Res. 40: 967–972). Herein, we report the construction of transgenic mice overexpressing the FABP5 gene encoding the epithelial fatty acid binding protein (E-FABP) in adipocytes, thereby allowing evaluation of the effects on lipolysis of increased FABP levels and of type specificity. In adipocytes from FABP5 transgenic mice, the total FABP protein level in the adipocyte was increased to 150% as compared to the wild type due to a 10-fold increase in the level of E-FABP and an unanticipated 2-fold down-regulation of the A-FABP. There were no significant differences in body weight, serum FFA, or fat pad mass between wild-type and FABP5 transgenic mice. Importantly, both basal and hormone-stimulated lipolysis increased in adipocytes from the FABP5 transgenic animals. The molecular composition of the fatty acid pool from either the intracellular compartment or that effluxed from the adipocyte was unaltered.These results demonstrate that there is a positive relationship between lipolysis and the total level of FABP but not between lipolysis and a specific FABP type. Fatty acid binding proteins (FAB2222251) are low-molecular-mass, soluble, intracellular lipid carriers. Previous studies on adipocytes from adipocyte fatty acid binding protein (A-FABP)-deficient mice have revealed that both basal and isoproterenol-stimulated lipolysis were markedly reduced (Coe et al. 1999. J. Lipid Res. 40: 967–972). Herein, we report the construction of transgenic mice overexpressing the FABP5 gene encoding the epithelial fatty acid binding protein (E-FABP) in adipocytes, thereby allowing evaluation of the effects on lipolysis of increased FABP levels and of type specificity. In adipocytes from FABP5 transgenic mice, the total FABP protein level in the adipocyte was increased to 150% as compared to the wild type due to a 10-fold increase in the level of E-FABP and an unanticipated 2-fold down-regulation of the A-FABP. There were no significant differences in body weight, serum FFA, or fat pad mass between wild-type and FABP5 transgenic mice. Importantly, both basal and hormone-stimulated lipolysis increased in adipocytes from the FABP5 transgenic animals. The molecular composition of the fatty acid pool from either the intracellular compartment or that effluxed from the adipocyte was unaltered. These results demonstrate that there is a positive relationship between lipolysis and the total level of FABP but not between lipolysis and a specific FABP type. Mammalian fatty acid binding proteins (FABPs) are expressed from a nine-member multi-gene family in tissues involved with active lipid metabolism (1Coe N.R. Bernlohr D.A. Physiological properties and functions of intracellular fatty acid-binding proteins.Biochim. Biophys. Acta. 1998; 1391: 287-306Google Scholar, 2Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Intracellular lipid-binding proteins and their genes.Annu. Rev. Nutr. 1997; 17: 277-303Google Scholar, 3Veerkamp J.H. Maatman R.G. Cytoplasmic fatty acid-binding proteins: their structure and genes.Prog. Lipid Res. 1995; 34: 17-52Google Scholar). Some cells contain a single member of the family, whereas others express multiple family members. Structural analyses have documented, despite considerable divergence of the primary sequence, that the FABPs fold as a conserved β-barrel forming an interior water-filled cavity into which the ligand noncovalently resides (4LaLonde J.M. Bernlohr D.A. Banaszak L.J. The up-and-down beta-barrel proteins.FASEB J. 1994; 8: 1240-1247Google Scholar). Typically, a single long-chain fatty acid or other hydrophobic molecule is bound within the cavity, although the liver FABP binds two ligands (5Haunerland N. Jagschies G. Schulenberg H. Spener F. Fatty-acid-binding proteins. Occurrence of two fatty-acid-binding proteins in bovine liver cytosol and their binding of fatty acids, cholesterol, and other lipophilic ligands.Hoppe Seylers Z. Physiol. Chem. 1984; 365: 365-376Google Scholar). Even though specific in vivo functions have remained elusive, FABPs have been thought to provide solubility and/or intracellular trafficking of long-chain fatty acids and other hydrophobic ligands between metabolic enzymes and/or membranes (1Coe N.R. Bernlohr D.A. Physiological properties and functions of intracellular fatty acid-binding proteins.Biochim. Biophys. Acta. 1998; 1391: 287-306Google Scholar, 3Veerkamp J.H. Maatman R.G. Cytoplasmic fatty acid-binding proteins: their structure and genes.Prog. Lipid Res. 1995; 34: 17-52Google Scholar, 6Hertzel A.V. Bernlohr D.A. The mammalian fatty acid-binding protein multigene family: molecular and genetic insights into function.Trends Endocrinol. Metab. 2000; 11: 175-180Google Scholar, 7Bernlohr D.A. Coe N.R. LiCata V.J. Fatty acid trafficking in the adipocyte.Semin. Cell Dev. Biol. 1999; 10: 43-49Google Scholar).Adipocytes play a dynamic role in lipid metabolism and homeostasis, including the traditional function of storing triacylglycerol during conditions of energy excess and mobilizing fatty acids for utilization by other tissues in times of energy depletion (8Bernlohr D.A. Simpson M.A. Adipose tissue and lipid metabolism.in: Biochemistry of Lipids, Lipoproteins and Membranes. Elsevier, Amsterdam1996: 257-281Google Scholar). Additionally, adipocytes secrete cytokines in response to various metabolic and hormonal signals indicating an alteration in the energy status of the organism. These secreted proteins can function in a paracrine, autocrine, or endocrine manner, thereby regulating overall body energy metabolism (9Shuldiner A.R. Yang R. Gong D.W. Resistin, obesity and insulin resistance-the emerging role of the adipocyte as an endocrine organ.N. Engl. J. Med. 2001; 345: 1345-1346Google Scholar, 10Kim S. Moustaid-Moussa N. Secretory, endocrine and autocrine/paracrine function of the adipocyte.J. Nutr. 2000; 130 (Suppl.): 3110-3115Google Scholar, 11Trayhurn P. Beattie J.H. Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ.Proc. Nutr. Soc. 2001; 60: 329-339Google Scholar, 12Wauters M. Considine R.V. Van Gaal L.F. Human leptin: from an adipocyte hormone to an endocrine mediator.Eur. J. Endocrinol. 2000; 143: 293-311Google Scholar). Two genes from the FABP family are expressed in adipocytes: FABP4 [encoding the major form termed adipocyte fatty acid binding protein (A-FABP), also called aP2 or ALBP (A-form)], as well as FABP5 [encoding a minor form termed epithelial fatty acid binding protein (E-FABP), also called mal-1 or KLBP (E-form)]. In the wild-type mouse, E-FABP is approximately 1% to 5% that of A-FABP in the adipocyte (13Coe N.R. Simpson M.A. Bernlohr D.A. Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels.J. Lipid Res. 1999; 40: 967-972Google Scholar) making the A/E protein ratio approximately 20:1. Both proteins bind fatty acids with comparable affinities as well as similar specificities (14Kane C.D. Coe N.R. Vanlandingham B. Krieg P. Bernlohr D.A. Expression, purification, and ligand-binding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding found overexpressed in neoplastic skin cells.Biochemistry. 1996; 35: 2894-2900Google Scholar, 15Bernlohr D.A. Coe N.R. Simpson M.A. Hertzel A.V. Regulation of gene expression in adipose cells by polyunsaturated fatty acids.Adv. Exp. Med. Biol. 1997; 422: 145-156Google Scholar, 16Simpson M.A. LiCata V.J. Ribarik-Coe N. Bernlohr D.A. Biochemical and biophysical analysis of the intracellular lipid binding proteins of adipocytes.Mol. Cell. Biochem. 1999; 192: 33-40Google Scholar). In addition to expression in adipocytes, A-FABP is expressed in macrophages, whereas E-FABP expression is more widespread, including lens, macrophage, retina, tongue, lung, brain, and skin, as well as a variety of epithelial and endothelial cells (6Hertzel A.V. Bernlohr D.A. The mammalian fatty acid-binding protein multigene family: molecular and genetic insights into function.Trends Endocrinol. Metab. 2000; 11: 175-180Google Scholar, 17De Leon M. Welcher A.A. Nahin R.H. Liu Y. Ruda M.A. Shooter E.M. Molina C.A. Fatty acid binding protein is induced in neurons of the dorsal root ganglia after peripheral nerve injury.J. Neurosci. Res. 1996; 44: 283-292Google Scholar, 18Jaworski C. Wistow G. LP2, a differentiation-associated lipid-binding protein expressed in bovine lens.Biochem. J. 1996; 320: 49-54Google Scholar, 19Krieg P. Feil S. Furstenberger G. Bowden G.T. Tumor-specific overexpression of a novel keratinocyte lipid-binding protein. Identification and characterization of a cloned sequence activated during multistage carcinogenesis in mouse skin.J. Biol. Chem. 1993; 268: 17362-17369Google Scholar, 20Owada Y. Yoshimoto T. Kondo H. Increased expression of the mRNA for brain- and skin-type but not heart-type fatty acid binding proteins following kainic acid systemic administration in the hippocampal glia of adult rats.Brain Res. Mol. Brain Res. 1996; 42: 156-160Google Scholar).Knockout mouse models of several members of this family have been developed with the goal of identifying metabolic phenotypes linked to functional differences in FABP biology (6Hertzel A.V. Bernlohr D.A. The mammalian fatty acid-binding protein multigene family: molecular and genetic insights into function.Trends Endocrinol. Metab. 2000; 11: 175-180Google Scholar, 21Scheja L. Makowski L. Uysal K.T. Wiesbrock S.M. Shimshek D.R. Meyers D.S. Morgan M. Parker R.A. Hotamisligil G.S. Altered insulin secretion associated with reduced lipolytic efficiency in aP2-/- mice.Diabetes. 1999; 48: 1987-1994Google Scholar, 22Binas B. Danneberg H. McWhir J. Mullins L. Clark A.J. Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization.FASEB J. 1999; 13: 805-812Google Scholar, 23Schaap F.G. Binas B. Danneberg H. van der Vusse G.J. Glatz J.F. Impaired long-chain fatty acid utilization by cardiac myocytes isolated from mice lacking the heart-type fatty acid binding protein gene.Circ. Res. 1999; 85: 329-337Google Scholar). Utilizing such a strategy, FABP4-disrupted mice exhibit reduced basal as well as isoproterenol-stimulated lipolysis in situ and in vivo, suggesting a role for the protein in facilitating fatty acid efflux from the adipocyte (13Coe N.R. Simpson M.A. Bernlohr D.A. Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels.J. Lipid Res. 1999; 40: 967-972Google Scholar, 21Scheja L. Makowski L. Uysal K.T. Wiesbrock S.M. Shimshek D.R. Meyers D.S. Morgan M. Parker R.A. Hotamisligil G.S. Altered insulin secretion associated with reduced lipolytic efficiency in aP2-/- mice.Diabetes. 1999; 48: 1987-1994Google Scholar). Consistent with this view, A-FABP has been shown to physically interact with hormone-sensitive lipase (HSL) and stimulate its activity (24Shen W.J. Liang Y. Hong R. Patel S. Natu V. Sridhar K. Jenkins A. Bernlohr D.A. Kraemer F.B. Characterization of the functional interaction of adipocyte lipid-binding protein with hormone-sensitive lipase.J. Biol. Chem. 2001; 276: 49443-49448Google Scholar, 25Shen W.J. Sridhar K. Bernlohr D.A. Kraemer F.B. Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein.Proc. Natl. Acad. Sci. USA. 1999; 96: 5528-5532Google Scholar). Interestingly, FABP4 null animals up-regulate the expression of the FABP5 gene in adipocytes, resulting in an animal with reduced total FABP level as well as a switch in FABP types relative to wild-type animals (13Coe N.R. Simpson M.A. Bernlohr D.A. Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels.J. Lipid Res. 1999; 40: 967-972Google Scholar, 26Hotamisligil G.S. Johnson R.S. Distel R.J. Ellis R. Papaioannou V.E. Spiegelman B.M. Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein.Science. 1996; 274: 1377-1379Google Scholar). As such, the reduced lipolysis in FABP4 null mice could be linked to the loss of the A-FABP, the up-regulation of the E-FABP, or a generalized reduction in the total FABP content.To further characterize the relationship between levels of FABPs as well as of the specificity the type of FABP to lipolysis, a transgenic mouse was generated that overexpresses the FABP5 gene in the adipocyte driven by the FABP4 promoter. Herein, we report that such animals exhibit an increased total FABP level (150% of wild type) due to an increased level of E-FABP and a surprising decrease in the level of A-FABP resulting in an A/E ratio of 0.4. Adipocytes from such FABP5 transgenic mice exhibited increased basal and isoproterenol-stimulated lipolysis, indicating that lipolysis is linked to total FABP content and not to a specific FABP type.MATERIALS AND METHODSChemicalsAdenosine deaminase, isoproterenol, BSA (fatty acid free), N6-(R-1-methyl-2-phenethyl) phenylisopropyl adenosine (PIA), dinonyl phthalate, and boron trifluoride were obtained from Sigma Chemicals, St. Louis, MO. Fatty acid standards for gas chromatography were obtained from Nucheck Prep, MN. Bond Elut-aminopropyl silica gel columns were obtained from Varian Sample Preparations, Harbor City, CA. ECF Western blotting kit was obtained from Amersham Biosciences, Inc., Piscataway, NJ. The colorimetric nonesterified fatty acid (NEFA) assay kit was obtained from Wako, Richmond, VA.AnimalsC57Bl/6J wild-type, FABP4 null mice, and FABP5 transgenic mice were fed a standard low-fat diet ad libitum. All mice were weaned at 3 weeks of age and experiments were performed on 12 to 20 week old animals. To avoid anesthetic-induced artifacts in lipolysis, the mice were killed by cervical dislocation and blood samples were obtained through a cardiac puncture prior to dissection of the epididymal fat pads. Serum samples were frozen at −20°C prior to further analysis. All procedures were reviewed and approved by the University of Minnesota Animal Care and Use Committee.FABP5 transgenic miceThe complete murine FABP5 structural gene (27Hertzel A.V. Bernlohr D.A. Cloning and chromosomal location of the murine keratinocyte lipid-binding protein gene.Gene. 1998; 221: 235-243Google Scholar), from the transcriptional start site through the poly A site, was cloned downstream of the FABP4 5.4 kb promoter/enhancer (28Graves R.A. Tontonoz P. Platt K.A. Ross S.R. Spiegelman B.M. Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.J. Cell. Biochem. 1992; 49: 219-224Google Scholar) using the following strategy. Site-directed mutagenesis (29Kunkel T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc. Natl. Acad. Sci. USA. 1985; 82: 488-492Google Scholar) was used to introduce a Sac II site upstream of the FABP4 promoter/enhancer as well as two Not I sites flanking the FABP5 structural gene. The FABP5 gene was cloned into the Not I site at the 3′ side of FABP4 in pSKIIaP2 (28Graves R.A. Tontonoz P. Platt K.A. Ross S.R. Spiegelman B.M. Identification of a fat cell enhancer: analysis of requirements for adipose tissue-specific gene expression.J. Cell. Biochem. 1992; 49: 219-224Google Scholar). Subsequently, the vector sequences were removed by digestion with Sac II, followed by gel purification and dialysis of the fragment corresponding to the promoter/enhancer and structural gene. The DNA was microinjected into fertilized murine C57Bl/6J oocytes, and embryos were reimplanted in the uterus of a surrogate mother. Potential founders were identified by isolation of total genomic DNA and digestion with XbaI, followed by Southern blot analysis with an FABP5 intron 1 probe. The probe hybridized to a 2.5 kb band from an FABP5 transgenic mouse, whereas the corresponding wild-type band would be >20 kb. Three FABP5 transgenic lines were identified and backcrossed a minimum of three times to separate out independent integration sites. Initial analysis indicated that all lines behaved equivalently, and one was chosen for further study.Protein levels of FABPsEpididymal fat pads were dissected, homogenized with a Brinkman Polytron in PBS [10 mM sodium phosphate buffer (pH 6.8) containing 167 mM NaCl, 1 ml/g tissue] containing protease inhibitors, and centrifuged at 100,000 g for one h to produce a soluble extract. Varying amounts of protein were separated by SDS-PAGE along with known amounts of purified A-FABP and E-FABP standards (prepared in this laboratory). The proteins were transferred to a polyvinylidene difluoride membrane and blocked with TBS [10 mM Tris-HCl, 150 mM NaCl (pH 8.0)] containing 0.1% Tween-20 and 0.1% BSA. Rabbit affinity-purified polyclonal antibodies [directed toward either A-FABP (1:10,000 dilution) or E-FABP (1:1,000 dilution) prepared in this laboratory] were incubated overnight at room temperature and washed three times, and a secondary fluorescein-conjugated antibody was incubated for 1 h. Following additional washes, the fluorescent signal was obtained on a Storm Trooper Storm 840 Densitometer and analyzed using NIH Image software. The FABP concentration was determined from the standard curve plotted for each protein within the linear range of the signal.Isolation of primary adipocytesMurine epididymal fat pads from three to seven mice were pooled, minced, and suspended in a Krebs Ringer HEPES (KRH) buffered solution [118 mM NaCl, 4.75 mM KCl, 1.2 mM KH2PO4, 2.44 mM MgSO4, 25 mM NaHCO3, 2.52 mM CaCl2, 2.0 mM glucose, 200 nM adenosine, and 25 mM HEPES (pH 7.4)] containing 1 mg/ml collagenase (CLS1, Worthington Biochemicals) and 20 mg/ml BSA (Type V) prewarmed to 37°C. Adipocytes were isolated by collagenase digestion at 37°C for 60 min with vigorous shaking. The adipocytes were recovered by centrifugation at 2,000 rpm for 10 min at room temperature and washed twice in BSA-free KRH buffer supplemented with 200 nM adenosine, and subsequently resuspended in KRH buffer containing 2.5% BSA. The viability of the cells was verified by trypan blue exclusion analysis, and cell number was quantitated using an improved hemacytometer (Neubauer, American Scientific Products) prior to analyses.Lipid extraction and separationIntracellular fatty acids were extracted by homogenization of individual fat pads in a chloroform-methanol-phosphate-buffered saline solution (2:1:1.5; v/v/v) using a Brinkman Polytron tissue homogenizer. The homogenates were centrifuged at 4,000 rpm for 10 min at 25°C to generate a phase separation. The organic layer was removed and the extraction was repeated. The organic layers were pooled and concentrated under nitrogen to 1 ml. To separate the lipid classes, a Bond Elut-aminopropyl silica gel column (500 mg) was used (30Kaluzny M.A. Duncan L.A. Merritt M.V. Epps D.E. Rapid separation of lipid classes in high yield and purity using bonded phase columns.J. Lipid Res. 1985; 26: 135-140Google Scholar). Briefly, the lipids were loaded onto the column and washed sequentially with chloroform-2-propanol (2:1; v/v) to elute the neutral lipids and diethyl ether containing 2% acetic acid (98:2; v/v) to elute the FFAs. Test extractions using radioactive samples indicated that >98% of the input FFAs were recovered in the diethyl ether elution. For serum FFA analysis, total lipids were extracted from the serum in chloroform-methanol-water (2:1:1.5; v/v/v) using a volume five times the sample volume. The samples were vortexed for 1 min and centrifuged at 2,500 rpm for 10 min. The organic layer was removed and the extraction repeated. The organic phases were pooled and lipid classes were separated as described above.Fatty acid analysisThe extracted FFAs were converted into methyl esters using 14% boron trifluoride in methanol. Fatty acid derivatives were separated by gas chromatography using an HP 5890 gas chromatograph (Agilent Technologies) equipped with a flame ionization detector and integrator. Chromatography was performed using an Omegawax 320 fused silica capillary column (30 m× 0.32 mm internal diameter, 0.25 μm film thickness; Supelco, Bellefonte, PA). Individual fatty acid derivatives were identified by comparison of their retention times to those of authentic purified fatty acid standards. The colorimetric NEFA assay was used to quantify the total nonesterified FFAs as per the manufacturer’s instructions.Analysis of adipocyte lipolysisApproximately 25,000 cells were incubated in a final volume of 500 μl in KRH buffer supplemented with 200 nM adenosine, adenosine deaminase (1 unit/ml) and 10 μM PIA. Lipolysis was stimulated by the addition of 100 μM isoproterenol for 30 min at 37°C with vigorous shaking. The reaction was stopped by the addition of dinonyl phthalate oil followed by a low-speed centrifugation to separate the adipocytes from the incubation media. The total as well as individual fatty acids effluxed was analyzed as previously described. The data represent the mean value from pools of at least three mice each (n = 5) analyzed in triplicate for each genotype.Analysis of primary adipocyte fatty acid uptakeApproximately 50,000 cells were incubated in 550 μl KRH buffer supplemented with 2 mM glucose. BSA and oleic acid were preincubated for 30 min at 37°C. Uptake was initiated by addition of BSA/oleic acid (final concentration 50 μM each; [3H]oleic acid:1 Ci/mol) to the cells, resulting in a free oleic acid concentration of 6.85 nM. Reactions were maintained at 37°C for 30 and 300 s in quadruplicate. The reactions were stopped by the addition of 5 ml ice-cold 0.2 mM phloretin with 0.1% BSA, followed by vacuum filtration on glass microfiber filters to separate the unincorporated fatty acids from the cells. The filters were washed three times with 5 ml cold phloretin/BSA solution and dried, and the amount of [3H]oleic acid in the cells was determined by direct scintillation counting.Statistical analysesAll values are expressed as mean ± SEM unless otherwise noted. Statistical significance was determined by the two-tailed Student’s t-test. P values < 0.05 are considered significant.RESULTSTo test the hypothesis that FABPs facilitate fatty acid efflux during lipolysis, a transgenic mouse was developed that expresses high levels of FABP5 in adipocytes. Because the complete FABP5 gene is relatively small, a transgene containing the 5.4 kb FABP4 promoter/enhancer driving adipocyte expression of the complete FABP5 gene (Fig. 1A)was constructed and injected into fertilized murine C57Bl/6J oocytes. Successful insertion of the transgene was confirmed by Southern blot analysis (Fig. 1B). Mice with the strongest band on the Southern blot were mated to wild-type C57Bl/6J mice, and their offspring were analyzed for the transgene. Backcrossing to wild type allowed maintenance of the transgene in the heterozygous state. Three lines were continued and backcrossed a minimum of three times to allow segregation of multiple insertion sites. Copy number was also determined by Southern blot analysis comparing a fragment from the wild-type FABP5 gene (single copy) to the transgene that indicated approximately 10 copies of the transgene. The FABP5 transgenic mice had no apparent defect in fertility or growth, with no significant differences in body weight, offspring sex ratio, or epididymal fat pad mass (Table 1). Interestingly, approximately 10% of the adipocytes from the FABP5 transgenic mice were larger than the wild-type cells. Analyses were performed on all three lines with similar results; data from one line is reported herein.TABLE 1Physical and metabolic parameters of wild-type and FABP5 transgenic miceWild-TypeTransgenicTransgene copy number—10 ± 1Body weight (g)28.17 ± 2.4127.92 ± 1.65Epididymal fat pad wt (g)0.39 ± 0.080.43 ± 0.04A-FABP (ng/μg)2.73 ± 0.541.26 ± 0.31aE-FABP (ng/μg)0.15 ± 0.023.22 ± 0.49bTotal FABP (ng/μg)2.88 ± 0.664.48 ± 0.80% Adipocytes with diameter:0–2,000 (microns)83682,001–4,00014244,001–6,00023>6,00015A-FABP, adipocyte fatty acid binding protein; E-FABP, epithelial fatty acid binding protein. The transgene copy number was determined by Southern blot analysis and compared to the intensity of a single-copy gene. Fatty acid binding protein (FABP) levels were determined by Western blot analysis using chemifluorescence, and the signals were compared to known concentrations of purified protein. Adipocyte diameter was determined by light phase microscopy. Analyses were done on individual mice/samples from a minimum of 10 mice. Statistical significance comparing wild-type to FABP5 transgenic mice according to Student's t-test.a P < 0.05.b P < 0.001. Open table in a new tab Western blot analyses were performed on adipocyte protein extracts to examine whether the transgene was expressing FABP5 in adipocytes (Fig. 1C). Total levels of A-FABP and E-FABP were calculated by comparison to standard curves of each protein (Fig. 1D) using homogenous protein and monospecific polyclonal antibodies, both prepared in this laboratory. In wild-type adipocytes, E-FABP levels are 0.15 ng/μg protein while A-FABP levels are 2.73 ng/μg protein, leading to a total FABP level of 2.88 ng/μg protein and an A/E ratio of approximately 20:1. In the adipocytes from FABP5 transgenic animals, E-FABP levels were up-regulated to 3.22 ng/μg protein while A-FABP levels were down-regulated to 1.26 ng/μg protein, leading to an overall 150% increase in FABP content and an A/E ratio of 0.4:1. This is in contrast to the FABP4 null mice, which express only 10% adipocyte FABP levels (13Coe N.R. Simpson M.A. Bernlohr D.A. Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels.J. Lipid Res. 1999; 40: 967-972Google Scholar).Although the fatty acid binding specificity and affinity of E-FABP and A-FABP are similar, they are not identical (2Bernlohr D.A. Simpson M.A. Hertzel A.V. Banaszak L.J. Intracellular lipid-binding proteins and their genes.Annu. Rev. Nutr. 1997; 17: 277-303Google Scholar, 14Kane C.D. Coe N.R. Vanlandingham B. Krieg P. Bernlohr D.A. Expression, purification, and ligand-binding analysis of recombinant keratinocyte lipid-binding protein (MAL-1), an intracellular lipid-binding found overexpressed in neoplastic skin cells.Biochemistry. 1996; 35: 2894-2900Google Scholar, 16Simpson M.A. LiCata V.J. Ribarik-Coe N. Bernlohr D.A. Biochemical and biophysical analysis of the intracellular lipid binding proteins of adipocytes.Mol. Cell. Biochem. 1999; 192: 33-40Google Scholar). To determine if the overexpression of E-FABP in the FABP5 transgenic mice alters the composition of intracellular FFAs (the sum of bound and unbound), gas chromatography analysis was performed on lipids extracted from the fat pads of wild-type and FABP5 transgenic mice. From the two genotypes, 14 different fatty acids were identified and analyzed, with relative mol% ranging from 0.1% to 30.1% (Table 2). The predominant fatty acids identified were long-chain 16 and 18 carbon atoms with zero to two double bonds (mainly C16:0, C18:1, and C18:2) that accounted for 94% to 97% of the total fatty acids identified. No major significant differences in the composition of intracellular fatty acids were noted between the two genotypes. These results suggest that despite a change in A/E ratio from 20 to 0.4, the composition of the total intracellular FFA pool remained unaltered.TABLE 2Intracellular fatty acid composition in wild-type and FABP5 transgenic miceFatty AcidWild-TypeFABP5 Transgenicmol%C14:01.3 ± 0.21.67 ± 0.3C14:11.7 ± 0.41.97 ± 0.3C16:023.97 ± 1.025.17 ± 0.4C16:15.87 ± 0.75.57 ± 0.3C18:05.77 ±1.76.27 ± 1.0C18:127.47 ± 0.927.37 ± 1.4C18:230.07 ± 1.828.37 ± 0.5C18:32.57 ± 0.12.47 ± 0.1C20:10.37 ± 0.00.37 ± 0.0C20:20.77 ± 0.30.87 ± 0.2C20:30.07 ± 0.00.17 ± 0.1C20:40.67 ± 0.20.57 ± 0.0C22:00.17 ± 0.10.27 ± 0.1C22:10.47 ± 0.20.57 ± 0.0FFAs were esterified to methyl esters and analyzed by gas chromatography. The analysis was repeated three times for each genotype, with a pool of at least three mice per experiment. Values are expressed as mean ± SEM. Statistical significance was not identified, because all P values were > 0.05. Open table in a new tab To determine if the serum concentration of FFAs or the serum composition was altered in FABP5 transgenic animals relative to wild-type, samples were collected and the fatty acids extracted and analyzed by gas chromatography. Levels of plasma FFA were indistinguishable among the three genotypes (Table 3). Likewise, no significant differences were observed in the serum composition of the FFAs (data not shown).TABLE 3Serum FFA levels in male miceFFAmMWild-type0.76 ± 0.11FABP4 null0.55 ± 0.06FABP5 transgenic0.81 ± 0.11Quantitation of serum FFAs in wild-type, FABP4 null, and FABP5 transgenic mice. Serum was collected and FFAs were measured using a nonesterified fatty acid (NEFA) Wako kit. Analysis was done on at least seven mice from each genotype. Data are expressed as mean ± SEM. Open table in a new tab Lipolysis is an adipocyte function critical to maintaining energy homeostasis. Previous results on primary adipocytes from the FABP4 null mice demonstrated a 50% reduction in basal and isoproterenol-stimulated lipolysis. To investigate the relationship between total FABP levels, the A/E ratio, and the process of lipolysis, primary adipocytes from wild-type and FA" @default.
• W2050337777 created "2016-06-24" @default.
• W2050337777 creator A5007281588 @default.
• W2050337777 creator A5044835954 @default.
• W2050337777 creator A5081338125 @default.
• W2050337777 date "2002-12-01" @default.
• W2050337777 modified "2023-10-17" @default.
• W2050337777 title "Increased lipolysis in transgenic animals overexpressing the epithelial fatty acid binding protein in adipose cells" @default.
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