Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation

Ex-FABP, extracellular fatty acid binding protein, is a 21 kDa lipocalin expressed in hypertrophic cartilage, muscle and heart during chick embryo development and in granulocytes. Ex-FABP synthesis was increased in chondrocyte and myoblast cultures by inflammatory agents (LPS; IL6) and repressed by antiinflammatory agents. Expression of Ex-FABP and specific gelatinases is paralleled in hypertrophic cartilage; LPS specifically induced high molecular weight gelatinase ( > 200 kDa). LPS-treated hypertrophic chondrocytes showed increased chemotactic activity for endothelial cells paralleled by increased expression of transferrin. A high amount of Ex-FABP was expressed in adult pathological cartilage both in dyschondroplastic and osteoarthritic chickens. Controls were negative. Ex-FABP could represent a stress protein physiologically expressed in tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions. We also suggest that during endochondral bone formation other responses characteristic of a local inflammatory status, such as gelatinase production and angiogenic factor secretion, are "physiologically" activated.     

Hypoxia-induced fatty acid transporter translocation increases fatty acid transport and contributes to lipid accumulation in the heart

Protein-mediated LCFA transport across plasma membranes is highly regulated by the fatty acid transporters FAT/CD36 and FABPpm. Physiologic stimuli (insulin stimulation, AMP kinase activation) induce the translocation of one or both transporters to the plasma membrane and increase the rate of LCFA transport. In the hypoxic/ischemic heart, intramyocardial lipid accumulation has been attributed to a reduced rate of fatty acid oxidation. However, since acute hypoxia (15 min) activates AMPK, we examined whether an increased accumulation of intramyocardial lipid during hypoxia was also attributable to an increased rate of LCFA uptake as a result AMPK-induced translocation of FAT/CD36 and FABPpm. In cardiac myocytes, hypoxia (15 min) induced the redistribution of FAT/CD36 from an intracellular pool (LDM) (-25%, P<0.05) to the plasma membranes (PM) (+54%, P<0.05). Hypoxia also induced an increase in FABPpm at the PM (+56%, P<0.05) and a concomitant FABPpm reduction in the LDM (-24%, P<0.05). Similarly, in intact, Langendorff perfused hearts, hypoxia induced the translocation of a both FAT/CD36 and FABPpm to the PM (+66% and +61%, respectively, P<0.05), with a concomitant decline in FAT/CD36 and FABPpm in the LDM (-24% and -23%, respectively, P<0.05). Importantly, the increased plasmalemmal content of these transporters was associated with increases in the initial rates of palmitate uptake into cardiac myocytes (+40%, P<0.05). Acute hypoxia also redirected palmitate into intracellular lipid pools, mainly to PL and TG (+48% and +28%, respectively, P<0.05), while fatty acid oxidation was reduced (-35%, P<0.05). Thus, our data indicate that the increased intracellular lipid accumulation in hypoxic hearts is attributable to both: (a) a reduced rate of fatty acid oxidation and (b) an increased rate of fatty acid transport into the heart, the latter being attributable to a hypoxia-induced translocation of fatty acid transporters.     

Reduction of extracellular potassium ferricyanide by transmembrane NADH: (acceptor) oxidoreductase of human erythrocytes

Reduction of extracellular ferricyanide by intact erythrocytes proceeds by a membrane bound, NADH-dependent reaction. It is depressed by a glycolysis inhibitor and a non penetrable sulfhydryl reagent, and activated by dehydroascorbate. Dehydroascorbate activation cannot be accounted for by release of reducing equivalents from the cells. It is concluded that the observed reaction is brought about by transmembrane NADH-acceptor oxidoreductase with donor binding at the inner and acceptor binding at the outer cell surface.     

Targeted lipidomics: fatty acid amides and pain modulation

Mass spectrometric approaches to the identification and quantification of lipid signalling molecules are reviewed. Fatty acid amides are an important new class of lipid signalling molecules which include oleamide, the endocannabinoid anandamide, the endovanilloid/endocannabinoid N-arachidonoyldopamine (NADA) and the endovanilloid N-oleoyldopamine (OLDA) among many others. This diverse group of endogenous compounds comprises combinations of acyl backbones coupled by an amide bond to any of a variety of different small polar molecules such as ethanolamine, various amino acids, and catecholamines. Many fatty acid amides appear to play a role in pain and inflammation. Targeted lipidomics of fatty acid amides aims to identify new members of this diverse class of compounds, of which only a few representative molecules have been characterized to date. This effort has been made feasible by advances in chromatography and mass spectrometry, which permits: (1) identification of compounds present in complex mixtures, (2) astronomical increases in sensitivity due to miniaturization of HPLC components, and (3) novel scanning modes that permit the identification of compounds exhibiting similar structural components. Insofar as lipid signalling molecules such as prostanoids, leukotrienes and endocannabinoids operate via G-protein coupled receptors (GPCR), it appears likely that many of the numerous lipids awaiting identification may serve as ligands for any of the greater than 150 orphan GPCRs.     

Inhibition of 5-aminolevulinic acid (ALA) dehydratase by undissociated levulinic acid during ALA extracellular formation by Rhodobacter sphaeroides

The inhibition of ALA dehydratase by levulinic acid during ALA extracellular formation of Rhodobacter sphaeroides correlated with the concentration of undissociated form of levulinic acid irrespective of culture pH. The inhibition constant, Ki, of intracellular ALA dehydratase by Dixon plots was 2.95 μM. Undissociated levulinic acid therefore functions as an inhibitor of ALA dehydratase. This revised version was published online in November 2006 with corrections to the Cover Date.     

Contribution of omega-3 fatty acid desaturase and 3-ketoacyl-ACP synthase II (KASII) genes in the modulation of glycerolipid fatty acid composition during cold acclimation in birch leaves

Temperate and boreal tree species respond to low positive temperatures (LT) or a shortening of the photoperiod (SD) by inducing cold acclimation. One of the metabolic consequences of cold acclimation is an increase in fatty acid (FA) desaturation in membrane lipids, which allows functional membrane fluidity to be maintained at LT. The molecular mechanisms of FA desaturation were investigated in leaves of birch seedlings (Betula pendula) during cold acclimation. Four genes involved in FA biosynthesis were isolated: a 3-ketoacyl-ACP synthase II gene (BpKASII) involved in the elongation of palmitoyl-ACP to stearoyl-ACP, and three omega-3 FA desaturase genes (BpFAD3, BpFAD7, and BpFAD8) involved in the desaturation of linoleic acid (18:2) to alpha-linolenic acid (18:3). BpFAD7 was the main omega-3 FAD gene expressed in birch leaves, and it was down-regulated by LT under SD conditions. LT induced the expression of BpFAD3 and BpFAD8 and a synchronous increase in 18:3 occurred in glycerolipids. Changes in the photoperiod did not affect the LT-induced increase in 18:3 in chloroplast lipids (MGDG, DGDG, PG), but it modulated the LT response detected in extra-chloroplastic lipids (PC, PE, PI, PS). A decrease in the proportion of the 16-carbon FAs in lipids occurred at LT, possibly in relation to the regulation of BpKASII expression at LT. These results suggest that LT affects the whole FA biosynthesis pathway. They support a co-ordinated action of microsomal (BpFAD3) and chloroplast enzymes (BpFAD7, BpFAD8) in determining the level of 18:3 in extra-chloroplastic membranes, and they highlight the importance of dynamic lipid trafficking.     

Enhanced synthesis and extracellular accumulation of hyaluronic acid during stimulation of quiescent human fibroblasts by mouse epidermal growth factor

The effect of mouse epidermal growth factor (mEGF) on the synthesis of glycosaminoglycans and glycoproteins by human fibroblasts has been studied. The addition of physiological concentrations (10^?9 M) of mEGF to quiescent cultures preincubated in the absence of serum was found to elicit an increased incorporation of ³H-glucosamine into the glycosaminoglycans and glycoproteins of both the cellular and extracellular fractions. Although the growth response to the factor, as measured by DNA replication, was minimal under these conditions as compared with the effect of serum, the mEGF-induced incorporation of glucosamine into these cellular constituents and into the extracellular glycoproteins was comparable to that elicited by serum shift-up. Serum, however, caused a significantly larger incorporation of glucoasmine into extracellular, acid-soluble glycosaminoglycans, which were shown to contain hyaluronic acid as the major component. As previously demonstrated, the growth response to mEGF can be enhanced several fold by an mEGF-binding arginine esterase, which is normally associated with the factor in vivo, and by ascorbate. The esterase was found to increase markedly the mEGF-induced incorporation of glucosamine into extracellular hyaluronic acid, while the addition of ascorbic acid did not significantly alter glucosamine incorporation.     

Increased expression of poly(ADP-ribose) polymerase-1 contributes to caspase-independent myocyte cell death during heart failure

Poly(ADP-ribose) polymerase-1 (PARP-1) plays a pivotal role in regulating genome stability, cell cycle progression, and cell survival. However, overactivation of PARP has been shown to contribute to cell death and organ failure in various stress-related disease conditions. In this study, we examined the role of PARP in the development and progression of cardiac hypertrophy. We measured the expression of PARP in mouse hearts with physiological (swimming exercise) and pathological (aortic banding) cardiac hypertrophy as well as in human heart samples taken at the time of transplantation. PARP levels were elevated both in swimming and banded mice hearts and demonstrated a linear positive correlation with the degree of cardiac hypertrophy. A dramatic increase (4-fold) of PARP occurred in 6-wk banded mice, accompanied by apparent signs of ventricular dilation and myocyte cell death. PARP levels were also elevated (2- to 3-fold) in human hearts with end-stage heart failure compared with controls. However, we found no evidence of caspase-mediated PARP cleavage in either mouse or human failing hearts. Overexpression of PARP in primary cultures of cardiac myocytes led to suppression of gene expression and robust myocyte cell death. Furthermore, data obtained from the analysis of PARP knockout mice revealed that these hearts produce an attenuated hypertrophic response to aortic banding compared with controls. Together, these results demonstrate a role for PARP in the onset and progression of cardiac hypertrophy and suggest that some events related to cardiac hypertrophy growth and progression to heart failure are mediated by a PARP-dependent mechanism.     

Improved production of long-chain fatty acid in Escherichia coli by an engineering elongation cycle during fatty acid synthesis (FAS) through genetic manipulation

The microbial biosynthesis of fatty acid of lipid metabolism, which can be used as precursors for the production of fuels of chemicals from renewable carbon sources, has attracted significant attention in recent years. The regulation of fatty acid biosynthesis pathways has been mainly studied in a model prokaryote, Escherichia coli. During the recent period, global regulation of fatty acid metabolic pathways has been demonstrated in another model prokaryote, Bacillus subtilis, as well as in Streptococcus pneumonia. The goal of this study was to increase the production of long-chain fatty acids by developing recombinant E. coli strains that were improved by an elongation cycle of fatty acid synthesis (FAS). The fabB, fabG, fabZ, and fabI genes, all homologous of E. coli, were induced to improve the enzymatic activities for the purpose of overexpressing components of the elongation cycle in the FAS pathway through metabolic engineering. The beta-oxoacyl-ACP synthase enzyme catalyzed the addition of acyl-ACP to malonyl-ACP to generate beta- oxoacyl-ACP. The enzyme encoded by the fabG gene converted beta-oxoacyl-ACP to beta-hydroxyacyl-ACP, the fabZ catalyzed the dehydration of beta-3-hydroxyacyl-ACP to trans-2-acyl-ACP, and the fabI gene converted trans-2- acyl-ACP to acyl-ACP for long-chain fatty acids. In vivo productivity of total lipids and fatty acids was analyzed to confirm the changes and effects of the inserted genes in E. coli. As a result, lipid was increased 2.16-fold higher and hexadecanoic acid was produced 2.77-fold higher in E. coli JES1030, one of the developed recombinants through this study, than those from the wild-type E. coli.     

The acyl-CoA synthetase "bubblegum" (lipidosin): further characterization and role in neuronal fatty acid beta-oxidation.

Acyl-CoA synthetases play a pivotal role in fatty acid metabolism, providing activated substrates for fatty acid catabolic and anabolic pathways. Acyl-CoA synthetases comprise numerous proteins with diverse substrate specificities, tissue expression patterns, and subcellular localizations, suggesting that each enzyme directs fatty acids toward a specific metabolic fate. We reported that hBG1, the human homolog of the acyl-CoA synthetase mutated in the Drosophila mutant "bubblegum," belongs to a previously unidentified enzyme family and is capable of activating both long- and very long-chain fatty acid substrates. We now report that when overexpressed, hBG1 can activate diverse saturated, monosaturated, and polyunsaturated fatty acids. Using in situ hybridization and immunohistochemistry, we detected expression of mBG1, the mouse homolog of hBG1, in cerebral cortical and cerebellar neurons and in steroidogenic cells of the adrenal gland, testis, and ovary. The expression pattern and ability of BG1 to activate very long-chain fatty acids implicates this enzyme in the pathogenesis of X-linked adrenoleukodystrophy. In neuron-derived Neuro2a cells, mBG1 co-sedimented with mitochondria and was found in small vesicular structures located in close proximity to mitochondria. RNA interference was used to decrease mBG1 expression in Neuro2a cells and led to a 30-35% decrease in activation and beta-oxidation of the long-chain fatty acid, palmitate. These results suggest that in Neuro2a cells, mBG1-activated long-chain fatty acids are directed toward mitochondrial degradation. mBG1 appears to play a minor role in very long-chain fatty acid activation in these cells, indicating that other acyl-CoA synthetases are necessary for very long-chain fatty acid metabolism in Neuro2a cells.     

Norepinephrine-induced 1,2-diacylglycerol accumulation and change in its fatty acid composition in the isolated perfused rat heart

Phosphoinositide hydrolysis is elicited by -adrenoceptor stimulation in the myocardium, resulting in the generation of 1,2-diacylglycerol by the direct activation of phospholipase C. However, the physiological role of 1,2-diacylglycerol accumulation in the heart has been largely unexplored. Therefore, we studied the effects of norepinephrine on the accumulation of 1,2-diacylglycerol and its fatty acid composition, as well as its function in isolated perfused rat hearts. A 30 min perfusion with norepinephrine following a stabilization period of 25 min caused increases of 68% and 57% in 1,2-diacylglycerol levels in the heart at 10^–6 M and 5 × 10^–6 M, respectively, compared to controls. Analysis of its fatty acid composition showed a significant elevation in the percentages of 18:2 and 20:4 although the absolute amounts of these increases in fatty acids were relatively low when compared to the elevation in the total amount of 1,2-diacylglycerol. The change in contractility was not consistently related to an increase in 1,2-diacylglycerol. These results indicate that increase in 1,2-diacylglycerol level in response to norepinephrine perfusion was accompanied by a change in fatty acid composition of 1,2-diacylglycerol.     

Cardiac norepinephrine release: modulation by ovariectomy and estrogen

Previously, we have demonstrated that in contrast to male rats, female rats do not show an age-related reduction of depolarization-elicited norepinephrine (NE) release from cardiac synaptosomes (resealed nerve terminals). These results suggest that sex hormones such as estrogen may modulate NE release from cardiac synaptosomes prepared from female rats. The present study was designed to test the hypotheses that long-term estrogen depletion, resulting from ovariectomy, and estrogen replacement alters depolarization-elicited NE release from cardiac synaptosomes. Female F344 rats were divided into two groups, one of which underwent bilateral ovariectomy, whereas the other underwent a sham operation. Three ovariectomized subgroups received daily injections of conjugated equine estrogens, delta8,9-dehydroestrone or 17 alpha-dihydroequilenin. Another ovariectomized control subgroup and the sham-operated animals received daily injections of vehicle. After 90 or 270 days of treatment, the animals were sacrificed. Cardiac synaptosomes were prepared from each heart, incubated with [(3)H]-NE, and used to evaluate NE release capacity by exposure to 50 mM K(+). The effectiveness of the ovariectomy and the estrogenic actions of the test compounds was confirmed by evaluating vaginal smears, determining uterine weights, and measuring serum luteinizing hormone (LH) concentrations. Ovariectomy (after both 90 and 270 days) significantly increased depolarization-induced NE release compared with sham-operated rats. Treatment with all three estrogenic preparations reduced NE release in ovariectomized rats to values similar to those observed in sham-operated animals. Interestingly, NE release rates from rats treated with conjugated estrogens for 270 but not 90 days were significantly below that observed in age-matched sham animals. These results demonstrate that estrogen modulates depolarization-elicited NE release from cardiac nerve terminals. Such modulation may represent a protective action by estrogen at the cardiac synapse.     

Reduction in adipocyte ATP by lipolytic agents: relation to intracellular free fatty acid accumulation

Epinephrine, norepinephrine, ACTH, and dibutyryl 3',5'-cyclic AMP reduced adipocyte ATP levels during 60 min incubation; glucose displayed a protective effect. The reduction in adipocyte ATP levels could not be attributed solely to: a direct hormone effect, deficiency in metabolic substrate, activation of adenyl cyclase with ATP consumption, loss of adenine nucleotide from the cell or loss of cells during incubation, lipolytic rate per se, or extracellular accumulation of FFA or glycerol. To determine whether intracellular FFA accumulation was a causative factor, intracellular FFA levels were measured during hormone-stimulated lipolysis. This was accomplished by using sucrose-U-(14)C as a marker for the extracellular space to correct for contamination of cells by extracellular albumin-bound FFA. These experiments showed that the fall in adipocyte ATP correlated with FFA saturation of medium albumin and progressive accumulation of FFA within the adipocyte. Furthermore, the protective effect of glucose noted above was associated with a marked reduction in intracellular FFA as compared to the extracellular FFA pool. On the basis of these studies, combined with those in the literature, it is concluded that in vitro effects of lipolytic agents on adipocyte ATP levels are the net result of imparied ATP synthesis (uncoupled oxidative phosphorylation) in the face of normal or augmented ATP consumption.     

Studies of the modulation of essential fatty acid metabolism by fatty acids in cultured neuroblastoma and glioma cells

In cultured neuroblastoma cells (N1E-115), the metabolism of the essential fatty acid, linoleic acid (18:2 (n-6)), to arachidonic acid (20:4(n-6)) can be altered by other fatty acids in a manner supporting a concerted action of the modulating fatty acid on the desaturation and chain elongation enzymes. In further examination of mechanisms involved, cultured glioma (C-6) or neuroblastoma-glioma hybrids (NG-108-15) cells showed similar patterns of activation by some fatty acids (e.g., 20:3(n-6) and 20:4(n-6)), and inhibition (e.g., 18:3(n-3) or 22:6(n-3)) or no effect (e.g., 18:1(n-9), 20:3(n-3)) by others. In contrast, only inhibition by 20:4(n-6) was seen in cultured HeLa cells, suggesting that the intracellular interactions may not be universal in all cell lines. For fatty acids that activate 20:4(n-6) formation, the lag observed when substrate and activator were administered simultaneously was eliminated by preincubation with activator. Maximal activation occurred within 4 h for neuroblastoma and 2 h for glioma; in each cell line activation declined steadily for 10 h after removal of the activator. Inhibition of protein synthesis did not alter activation. As 98% of the fatty acid incorporated was esterified to triacylglycerol or phospholipid and only the triacylglycerol mass expanded, several manipulations to potentially alter the flow of acyl chains between these lipid pools were evaluated using dual-label and pulse-chase experiments. Results suggested that competition between 18:2(n-6) utilization for esterification to phospholipid and the desaturation-chain elongation sequence as well as a more direct and specific interaction of certain fatty acids with the enzymes may influence 20:4(n-6) formation. A model to explain these observations is discussed.