Porcine P2 myelin protein primary structure and bound fatty acids determined by mass spectrometry.

Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain-Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-A crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.

Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells.

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported. Furthermore, a decrease in fatty acid incorporation has been unexpectedly obtained when I-FABP reached 0. 72% of cytosolic proteins in fibroblasts (Prows et al. 1997. Arch. Biochem. Biophys. 340: 135). In the present study, the effect of a high level of I-FABP similar to amounts present in the small intestine was investigated in the human colon adenocarcinoma cell line, Caco-2. After transfection with human I-FABP cDNA, a clone expressing 1.5% I-FABP and unchanged level of liver FABP was selected. These cells, which had a lower rate of proliferation as compared with mock-transfected cells, developed the typical morphological characteristics of differentiated enterocytes. Incubation of differentiated cells with [(14)C]palmitate showed a 34% reduction (P < 0.01) of fatty acid incorporation, whereas the relative distribution of radiolabel into triglycerides was not affected. A nonsignificant 21% reduction of fatty acid incorporation was observed with another clone expressing 10-fold less I-FABP. In conclusion, a high level of I-FABP expressed in a differentiated enterocyte model inhibited fatty acid incorporation, by a mechanism which remains to be defined.

Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in human transitional bladder cancer and its role in inducing cell death.

The present study examined the expression and role of the thiazolidinedione (TZD)-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARgamma), in human bladder cancers. In situ hybridization shows that PPARgamma mRNA is highly expressed in all human transitional epithelial cell cancers (TCCa's) studied (n=11). PPARgamma was also expressed in five TCCa cell lines as determined by RNase protection assays and immunoblot. Retinoid X receptor alpha (RXRalpha), a 9-cis-retinoic acid stimulated (9-cis-RA) heterodimeric partner of PPARgamma, was also co-expressed in all TCCa tissues and cell lines. Treatment of the T24 bladder cancer cells with the TZD PPARgamma agonist troglitazone, dramatically inhibited 3H-thymidine incorporation and induced cell death. Addition of the RXRalpha ligands, 9-cis-RA or LG100268, sensitized T24 bladder cancer cells to the lethal effect of troglitazone and two other PPAR- activators, ciglitazone and 15-deoxy-delta(12,14)-PGJ2 (15dPGJ(2)). Troglitazone treatment increased expression of two cyclin-dependent kinase inhibitors, p21(WAF1/CIP1) and p16(INK4), and reduced cyclin D1 expression, consistent with G1 arrest. Troglitazone also induced an endogenous PPARgamma target gene in T24 cells, adipocyte-type fatty acid binding protein (A-FABP), the expression of which correlates with bladder cancer differentiation. In situ hybridization shows that A-FABP expression is localized to normal uroepithelial cells as well as some TCCa's. Taken together, these results demonstrate that PPARgamma is expressed in human TCCa where it may play a role in regulating TCCa differentiation and survival, thereby providing a potential target for therapy of uroepithelial cancers.

Bovine epidermal fatty acid-binding protein: determination of ligand specificity and cellular localization in retina and testis.

The fatty acid-binding protein (FABP) family consists of small, cytosolic proteins believed to be involved in the uptake, transport, and solubilization of their hydrophobic ligands. Members of this family have highly conserved sequences and tertiary structures. Using an antibody against testis lipid-binding protein, a member of the FABP family, a protein was identified from bovine retina and testis that coeluted with exogenously added docosahexaenoic acid during purification. Amino acid sequencing and subsequent isolation of its cDNA revealed it to be nearly identical to a bovine protein expressed in the differentiating lens and to be the likely bovine homologue of the human epidermal fatty acid-binding protein (E-FABP). From quantitative Western blot analysis, it was estimated that bovine E-FABP comprised 0.9%, 0.1%, and 2.4% of retina, testis, and lens cytosolic proteins, respectively. Binding studies using the fluorescent probe ADIFAB indicated that this protein bound fatty acids of differing levels of saturation with relatively high affinities. Kd values ranged from 27 to 97 nM. In addition, the protein was immunolocalized to the Müller cells in the retina as well as to Sertoli cells in the testis. The location of bovine E-FABP in cells known to be supportive to other cell types in their tissues and the ability of E-FABP to bind a variety of fatty acids with similar affinities indicate that it may be involved in the uptake and transport of fatty acids essential for the nourishment of the surrounding cell types.

Transfection of L6 myoblasts with adipocyte fatty acid-binding protein cDNA does not affect fatty acid uptake but disturbs lipid metabolism and fusion.

We studied the involvement of fatty acid-binding protein (FABP) in growth, differentiation and fatty acid metabolism of muscle cells by lipofection of rat L6 myoblasts with rat heart (H) FABP cDNA or with rat adipocyte (A) FABP cDNA in a eukaryotic expression vector which contained a puromycin acetyltransferase cassette. Stable transfectants showed integration into the genome for all constructs and type-specific overexpression at the mRNA and protein level for the clones with H-FABP and A-FABP cDNA constructs. The rate of proliferation of myoblasts transfected with rat A-FABP cDNA was 2-fold higher compared with all other transfected cells. In addition, these myoblasts showed disturbed fusion and differentiation, as assessed by morphological examination and creatine kinase activity. Uptake rates of palmitate were equal for all clone types, in spite of different FABP content and composition. Palmitate oxidation over a 3 h period was similar in all clones from growth medium. After being cultured in differentiation medium, mock- and H-FABP-cDNA-transfected cells showed a lower fatty acid-oxidation rate, in contrast with A-FABP-cDNA-transfected clones. The ratio of [14C]palmitic acid incorporation into phosphatidylcholine and phosphatidylethanolamine of A-FABP-cDNA-transfected clones changed in the opposite direction in differentiation medium from that of mock- and H-FABP-cDNA-transfected clones. In conclusion, transfection of L6 myoblasts with A-FABP cDNA does not affect H-FABP content and fatty acid uptake, but changes fatty acid metabolism. The latter changes may be related to the observed fusion defect.

Inhibition of lipid absorption as an approach to the treatment of obesity.

A reduction in fat intake may be achieved by making educated choices to reduce total calorie intake, to consume a lower quantity of total fats, or to modify the ratio of saturated-to-polyunsaturated lipids. Leptin agonists or NPY or CCK antagonists may prove to be useful to diminish appetite and thereby reduce the total intake of food. But eating has such cultural, social, and hedonistic attributes that such a single-pronged approach is unlikely to be successful. The use of fat substitutes may prove to be popular to provide a wide range of snack food options, but these are likely to be of minimal use in weight reduction programs because of their distribution of additives in only a limited number of foods. The inhibitors of lipid digestion will be modestly successful in the short term; their long-term success will be influenced by gastrointestinal adverse effects and the need to consume fat-soluble vitamin supplements to prevent the development of fat-soluble vitamin deficiencies. The inhibition of lipid absorption is an attractive targeted approach for the treatment of obesity, since this would reduce the uptake of visible as well as invisible fats, which would potentially offer convenient dosing, and could also be a means to inhibit secondarily the uptake of carbohydrate calories.

Localization and regulation of the putative membrane fatty-acid transporter (FAT) in the small intestine. Comparison with fatty acid-binding proteins (FABP).

The expression of the putative membrane fatty-acid transporter (FAT) was investigated in the small intestine. The FAT mRNA level was higher in the jejunum than in the duodenum and was lower in the ileum, as observed for cytosolic fatty-acid-binding proteins (FABP) expressed in this tissue. No FAT transcript was found in the stomach or colon. FAT mRNA was constitutively expressed in the epithelial cells located in the upper two thirds of villi, while it was undetectable in the crypt cells and submucosal cells. In jejunal mucosa, immunochemical studies showed that FAT protein was limited to the brush border of enterocytes. No fluorescence was found in the goblet cells. To determine whether FAT responded to changes in fat intake, as reported for FABP, the effect of two high-fat diets, which essentially contained either medium-chain fatty acids or long-chain fatty acids (sunflower-oil diet), was investigated. The sunflower-oil diet greatly increased FAT mRNA abundance throughout the small intestine. In contrast, a weak effect of medium-chain fatty acids was observed only in the jejunum. As found for FABP expression, treatment with the hypolipidemic drug bezafibrate affected FAT expression. These data demonstrate that FAT and FABP are co-expressed in enterocytes, as has been shown in adipocytes, myocytes and mammary cells. The data suggest that these membrane and cytosolic proteins might have complementary functions during dietary-fat absorption.

A polymorphism in the human intestinal fatty acid binding protein alters fatty acid transport across Caco-2 cells.

The human intestinal fatty acid binding protein (IFABP) binds long-chain fatty acids in vitro, but its intracellular function has remained speculative. A polymorphism in the gene that encodes IFABP results in an alanine (Ala54) to threonine (Thr54) substitution at codon 54 that alters the in vitro binding affinity of the protein for long-chain fatty acids. To identify potential functional variability between Ala54 and Thr54 IFABP, we established permanently transfected Caco-2 cell lines that express either Ala54 or Thr54 IFABP. We found that Caco-2 cells expressing Thr54 IFABP transport long-chain fatty acids and secrete triglycerides to a greater degree than Caco-2 cells expressing Ala54 IFABP. These results provide the first demonstration that IFABP participates in the intracellular transport of long-chain fatty acids. In addition, the observed increase in transport of fatty acids across cells expressing Thr54 IFABP suggests a plausible physiologic mechanism for our prior observation that Pima Indians with a Thr54 IFABP genotype have increased post-absorptive lipid oxidation rates and are more insulin-resistant than Pimas with a Ala54 IFABP genotype.

Affinity of fatty acid for (r)rat intestinal fatty acid binding protein:further examination.

The enhancement of the fluorescence quantum yield of 1,8-anilinonaphthalenesulfonic acid (ANS) upon binding to intestinal fatty acid protein (I-FABP) was exploited to devise an assay for free I-FABP. With this assay, we monitored the competition for free I-FABP between ANS and fatty acids and thereby extracted values for the dissociation constants (K(FA)) of fatty acids for I-FABP. We obtained these constants for the I-FABP ligands oleic acid, arachidonic acid, and palmitic acid. In addition, we measured the dependence of K(FA) for oleic acid upon temperature and at two pH values. From these data, we calculate the van't Hoff enthalpy of oleic acid binding. This enthalpy is compared with the enthalpies of binding obtained directly from titration calorimetry. Our experiments with the fluorescence-based assay generate values of K(FA) which disagree with older values obtained from calorimetry and other methods. Our own calorimetric data were analyzed with a view to improving the technique involved in subtraction of a "reference" dilution of the ligand into solution in the absence of the protein. By this maneuver, we obtained "corrected" titrations which could be fitted to values of K(FA) more in agreement with the values we determined via the fluorescence-based assay than wer the older literature values. Our new values for K(FA) also agree substantially with values derived using a complementary assay technique, one measuring the concentration of free fatty acid, that has recently been developed by Richiere et al [Richiere et al. (1995) J. Biol. Chem. 270, 15076-15084]. We compare the values of delta H degrees, delta S degrees, and delta C(p)degrees for fatty acid binding we have obtained in this work with those we found in earlier work with ANS binding to I-FABP [Kirk et al. (1996) Biophys. J. 70, 69-83]. Our interpretation of the origin of the thermodynamic changes for ANS binding in our earlier work is here substantiated and extended to include an evaluation in physical terms of the interaction of I-FABP with fatty acids.

Decreased glutathione peroxidase activity in mice in response to nafenopin is caused by changes in selenium metabolism.

The activity of selenium-dependent glutathione peroxidase is known to be reduced in the liver of both rats and mice after exposure to nafenopin, as well as other peroxisome proliferators. The mechanism for this down-regulation is not known, but might involve changes in incorporation of selenium into selenoproteins. In this paper we show that both incorporation of selenium into selenoproteins and the level of selenium in liver is reduced in mice treated with nafenopin. The activity of selenium dependent glutathione peroxidase (GPx), as well as incorporation of selenium into its 23 kD subunit were found to be decreased. Contrary to what might have been expected, the decreased GPx activity was detected concomitantly with a slight increase in mRNA levels after 10 days of treatment, while a small decrease in mRNA levels was detected in treated animals after 26 weeks, together with the decrease in GPx-activity. Incorporation of selenium into liver fatty acid binding protein (L-FABP) was also decreased, even though large increases in protein and mRNA levels were detected. Taken together these data suggest that the decrease in GPx-activity in response to nafenopin is due to post-transcriptional mechanisms, involving changes in selenium metabolism.

3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate concentrations to induce gene amplification. Stably transfected clones were characterized by Southern blotting; those with highest copy numbers of pFR400 alone (pFR400) or pFR400 and pMAAT2 (pFR400/pMAAT2) were expanded for further study. [3H]Oleate uptake was measured in medium containing 500 microM bovine serum albumin and 125-1000 microM total oleate (unbound oleate, 18-420 nM) and consisted of saturable and nonsaturable components. pFR400/pMAAT2 cells exhibited no increase in the rate constant for nonsaturable oleate uptake or in the uptake rate of [14C]octanoate under any conditions. By contrast, Vmax (fmol/sec per 50,000 cells) of the saturable oleate uptake component increased 3.5-fold in pFR400/pMAAT2 cells compared to pFR400, with a further 3.2-fold increase in the presence of Zn2+. Zn2+ had no effect in pFR400 controls (P > 0.5). The overall increase in Vmax between pFR400 and pFR400/pMAAT2 in the presence of Zn2+ was 10.4-fold (P < 0.01) and was highly correlated (r = 0.99) with expression of FABPpm in plasma membranes as determined by Western blotting. Neither untransfected 3T3 nor pFR400 cells expressed cell surface FABPpm detectable by immunofluorescence. By contrast, plasma membrane immunofluorescence was detected in pFR400/pMAAT2 cells, especially if cultured in 100 microM Zn2+. The data support the dual hypotheses that mAspAT and FABPpm are identical and mediate saturable long-chain free fatty acid uptake.

Influence of liposomes rich in unsaturated or saturated fatty acids on the growth of human xenotransplanted mammary carcinomas and on the levels of heart type fatty acid binding protein.

A panel of 4 human mammary carcinomas passaged in nude mice were subjected to intraperitoneal application of cholesterol-free liposomes enriched with linoleic (unsaturated fatty acid) or stearic acid (saturated fatty acid). The liposomes were examined with regard to their influence on the tumor growth and level of heart type fatty acid binding protein (FABP). Liposomes with different fatty acid composition influenced the growth of mammary carcinomas 3366, BO, 4000 and 4151 in distinct ways. Liposomes with a high content of stearic acid significantly inhibited the growth of mammary carcinomas 3366 and BO, whereas mammary carcinomas 4000 and 4151 were not affected. The growth of mammary carcinoma 3366 was moderately increased after supplementation of liposomes rich in linoleic acid, the tumor BO was significantly inhibited and the growth of MaCa 4000 and 4151 was unchanged. Liposome treatment led to a significant increase in heart type FABP in mammary carcinomas 3366 and BO regardless of whether the animals were treated with liposomes rich in stearic or linoleic acid. Such significant changes of FABP level could not be observed in mammary carcinomas 4000 or 4151. We suggest that the lipid-mediated growth modulation seems to be dependent on an increase of heart type FABPs in these tumor models.