Triacylglycerol synthesis in isolated fat cells. An effect of insulin on microsomal fatty acid coenzyme A ligase activity.

Fatty acid CoA ligase (AMP) (EC 6.2.1.3) specific activity was increased approximately 2-fold in microsomes prepared from isolated rat fat cells incubated with 400 microunits of insulin/ml (2.9 nM) for 45 to 60 min compared to paired controls using an assay based on the conversion of [3H]oleic acid to [3H]oleoyl-CoA. Similar insulin-dependent increases in microsomal fatty acid CoA ligase specific activities were observed using an assay based on the conversion of [3H]CoA to fatty acyl-[3H]CoA. Fatty acid CoA ligase activity was predominately (about 80%) associated with the microsomal fraction. The insulin-dependent increase in microsomal fatty acid CoA ligase specific activity was maximal in 2 to 5 min at 400 microunits/ml. At 10 min, 80 to 100 microunits of insulin/ml caused a maximal increase in fatty acid CoA ligase specific activity. Similar apparent Km values for ATP, CoA, and fatty acid were observed for fatty acid CoA ligase activity in microsomal preparations from control and insulin-exposed cells. These data suggest that fatty acid CoA ligase activity is regulated in adipose tissue by insulin. Such regulation may serve to promote the capture of fatty acid and thereby, triacylglycerol synthesis in adipose tissue.     

Fatty acid alcohol ester-synthesizing activity of lipoprotein lipase

The fatty acid alcohol ester-synthesizing activity of lipoprotein lipase (LPL) was characterized using bovine milk LPL. Synthesizing activities were determined in an aqueous medium using oleic acid or trioleylglycerol as the acyl donor and equimolar amounts of long-chain alcohols as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with LPL, palmityl oleate was synthesized, in a time- and dose-dependent manner. Apo-very low density lipoprotein (apoVLDL) stimulated LPL-catalyzed palmityl oleate synthesis. The apparent equilibrium ratio of fatty acid alcohol ester/oleic acid was estimated using a high concentration of LPL and a long (20 h) incubation period. The equilibrium ratio was affected by the incubation pH and the alcohol chain length. When the incubation pH was below pH 7.0 and long chain fatty acyl alcohols were used as substrates, the fatty acid alcohol ester/free fatty acid equilibrium ratio favored ester formation, with an apparent equilibrium ratio of fatty acid alcohol ester/fatty acid of about 0.9/0.1. The equilibrium ratio decreased sharply at alkaline pH (above pH 8.0). The ratio also decreased when fatty alcohols with acyl chains shorter than dodecanol were used. When a trioleoylglycerol/fatty acyl alcohol emulsion was incubated with LPL, fatty acid alcohol esters were synthesized in a dose- and time-dependent fashion. Fatty acid alcohol esters were easily synthesized from trioleoylglycerol when fatty alcohols with acyl chains longer than dodecanol were used, but synthesis was decreased with fatty alcohols with acyl chain lengths shorter than decanol, and little synthesizing activity was detected with shorter-chain fatty alcohols such as butanol or ethanol.     

Plasma fatty acid metabolic profiling and biomarkers of type 2 diabetes mellitus based on GC/MS and PLS-LDA

Metabolic profiling has increasingly been used as a probe in disease diagnosis and pharmacological analysis. Herein, plasma fatty acid metabolic profiling including non-esterified fatty acid (NEFA) and esterified fatty acid (EFA) was investigated using gas chromatography/mass spectrometry (GC/MS) followed by multivariate statistical analysis. Partial least squares-linear discrimination analysis (PLS-LDA) model was established and validated to pattern discrimination between type 2 diabetic mellitus (DM-2) patients and health controls, and to extract novel biomarker information. Furthermore, the PLS-LDA model visually represented the alterations of NEFA metabolic profiles of diabetic patients with abdominal obesity in the treated process with rosiglitazone. The GC/MS-PLS-LDA analysis allowed comprehensive detection of plasma fatty acid, enabling fatty acid metabolic characterization of DM-2 patients, which included biomarkers different from health controls and dynamic change of NEFA profiles of patients after treated with medicine. This method might be a complement or an alternative to pathogenesis and pharmacodynamics research.     

A live-cell high-throughput screening assay for identification of fatty acid uptake inhibitors

We developed a live-cell high-throughput assay system using the baker's yeast Saccharomyces cerevisiae to screen for chemical compounds that will inhibit fatty acid uptake. The target for the inhibitors is a mammalian fatty acid transport protein (mmFATP2), which is involved in the fatty acid transport and activation pathway. The mmFATP2 was expressed in a S. cerevisiae mutant strain deficient in Fat1p-dependent fatty acid uptake and reduced in long-chain fatty acid activation, fat1Deltafaa1Delta. To detect fatty acid import, a fluorescent fatty acid analog, 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (C1-BODIPY-C12), was incubated with cells expressing FATP2 in a 96-well plate. The mmFATP2-dependent C1-BODIPY-C12 uptake was monitored by measuring intracellular C1-BODIPY-C12 fluorescence on a microtiter plate reader, whereas extracellular fluorescence was quenched by a cell viability dye, trypan blue. Using this high-throughput screening method, we demonstrate that the uptake of the fluorescent fatty acid ligand was effectively competed by the natural fatty acid oleate. Inhibition of uptake was also demonstrated to occur when cells were pretreated with sodium azide or Triacsin C. This yeast live-cell-based assay is rapid to execute, inexpensive to implement, and has adequate sensitivity for high-throughput screening. The assay basis and limitations are discussed.     

Ca2+ translocation across liposomal membranes enhanced by unsaturated long-chain fatty acids

The putative ionophoretic action of phosphatidic acid or arachidonic acid metabolites for Ca2+ has offered an attractive explanation for stimulation-coupled mobilization of cytoplasmic Ca2+. We have examined the effects of Ca2+ ionophore and long-chain unsaturated fatty acids on the translocation of Ca2+ across the liposomal membrane by using Quin II-entrapped liposomes, a sensitive assay system for ionophoresis of Ca2+. A23187 increased Quin II fluorescence intensity corresponding to the translocation of Ca2+ into liposomes. Similar translocation was observed with unsaturated long-chain fatty acids but not with saturated fatty acids. Thus, when phospholipases of cell membrane are activated by certain stimuli, unsaturated long-chain fatty acids are liberated and might mediate the mobilization of cytoplasmic Ca2+.     

Improved detection of polyunsaturated fatty acids as phenacyl esters using liquid chromatography-ion trap mass spectrometry

The fatty acid composition of Shewanella pealeana was determined by the analysis of fatty acid methyl esters via gas chromatography-mass spectrometry (GC-MS) and fatty acid 2-oxo-phenylethyl esters via high-performance liquid chromatography-mass spectrometry (LC-MS) combined with ultra violet (UV) detection. There was good agreement between the percentage composition of components determined by GC-MS and LC-UV analyses. However, LC-MS analysis using Atmospheric Pressure Chemical Ionization (APCI) demonstrated dramatically enhanced detection of unsaturated fatty acid 2-oxo-phenylethyl esters. The degree of enhancement was proportional to the degree of unsaturation. Tests with a pure polyunsaturated fatty acid (PUFA) standard gave an absolute detection limit in full scan mode of 200 pg. In samples, the selectivity of MS over UV gave a significantly lower detection limit due to lack of chemical interferences. In 'Selected Reaction Monitoring' (SRM) mode, the detection limit was 5 pg. This was essentially independent of whether the sample is a standard or complex mixture of fatty acids. Tandem mass spectrometry was used to support structural information and to enhance the ability to target specific fatty acids. Several PUFAs which were not evident from GC-MS analysis were detected and identified by APCI LC-MS, including some rare or novel PUFAs from S. pealeana and a menhaden oil standard. Detailed analysis of bacterial fatty acid composition by either GC-MS or APCI LC-MS is highly preferable to analysis systems based solely on retention time identification.     

Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 micrograms/mg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification.     

Demonstration of diet-induced decoupling of fatty acid and cholesterol synthesis by combining gene expression array and 2H2O quantification

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate-free diet. At the end of the dietary intervention, the two groups received (2)H(2)O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a custom-designed pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydrate-free diet. In support of these findings, (2)H(2)O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered.     

Interaction between free fatty acids and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.     

Assay of fatty acid synthetase by mass fragmentography using [13C]malonyl-CoA

A new assay method for fatty acid synthetase using mass fragmentography was described. [2-13C]Malonyl-CoA was chemically synthesized from [2-13C]malonic acid and used as a substrate. The newly synthesized fatty acids were quantitated with a GC-MS instrument after methyl esterification. Monitoring of molecular ions of the newly synthesized fatty acids enabled us to determine the absolute amounts with heptadecanoic acid as an internal standard. Multiple products (14 : 0, 16 : 0, and 18 : 0) were measured individually. Using this technique, we obtained information about production profiles such as that of chain length against incubation temperature and about malonyl-CoA decarboxylation activity in enzyme preparations, and we also confirmed the presence of malonyl-CoA decarboxylation activity even in purified fatty acid synthetase from guinea pig Harderian gland. Compared with the conventional assay methods (spectrophotometric and radioisotopic), this method was more reliable and useful.     

Calcium modulates fatty acid dynamics in rat liver plasma membranes

Modulation of free fatty acid binding in isolated rat liver plasma membranes was evaluated using the fluorescent fatty acids trans-parinaric and cis-parinaric acid as analogues for saturated and unsaturated fatty acids, respectively. Binding of trans-parinarate but not cis-parinarate was inhibited by physiological levels of Ca2+. The effect was reversed by addition of excess EGTA. Calcium decreased the aqueous to lipid partition coefficient, Kp, of trans-parinaric acid for liver plasma membranes while increasing the Kp for cis-parinaric acid. In addition, Ca2+ also altered the fluorescence lifetime, the quantum yield, and the relative partitioning of trans-parinaric and cis-parinaric acid into fluid and solid phases. Calcium and EGTA did not affect the binding of 1,6-diphenyl-1,3,5-hexatriene. The effect of Ca2+ on the liver plasma membrane structure was to increase the rigidity of the membrane, primarily the solid domain. The fluorescence polarization of trans-parinarate, cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene at 24 degrees C in liver plasma membranes in the absence of Ca2+ was 0.295 +/- 0.008, 0.253 +/- 0.007, and 0.284 +/- 0.005, respectively. Calcium (2.4 mM) increased the polarization of these probe molecules in liver plasma membranes by 8-10%. EGTA (3.4 mM) reversed or abolished the increase in polarization. Thus, the fluorescent fatty acids trans-parinarate and cis-parinarate may be used to monitor fatty acid binding by isolated membranes, to evaluate factors such as Ca2+ which modulate fatty acid binding, and to investigate the microenvironment in which the fatty acids residue. The data suggest that Ca2+ may be an important regulator of fatty acid uptake by the liver plasma membrane, and thereby interact with intermediary metabolism of lipids at a step not involving lipolytic or synthetic enzymes.     

Phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions. Regulation of fatty acid positional specificity.

The positional and fatty acid specificity of phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions was studied by using acylcarnitines, CoA and an excess of carnitine palmitoyltransferase (EC 2.3.1.21) as the source of acyl-CoA. In the mitochondria, the preference for palmitic acid at the 1-position is increased at high acyl-CoA concentrations, whereas it is decreased in the microsomal fraction. There was no change in the fatty acid specificity at the 2-position with different acyl-CoA concentrations in any of the factions. The preference in mitochondria for linoleic acid at the 2-position is strongly increased at high concentrations of lysophosphatidic acid.     

Polyunsaturated fatty acid supplements modulate mast cell membrane microdomain composition

In the present study, the lipid raft composition of a canine mastocytoma cell line (C2) was analyzed. Lipid rafts were well separated from non-raft plasma membranes using a detergent-free isolation technique. To study the influence of n-3 and n-6 polyunsaturated fatty acids (PUFA) on raft fatty acid composition in comparison to non-raft cell membrane, C2 were supplemented with one of the following: α-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, linoleic acid or arachidonic acid. Enrichment of the culture medium with a specific PUFA resulted in an increase in the content of this fatty acid both in rafts and non-raft membranes. Contents of cholesterol and protein were found not to be affected by the changes in the fatty acid profiles. In conclusion, our data provide strong evidence that PUFA modulate lipid composition and physiological properties of membrane micro domains of mast cells which in turn may have effects on mast cell function.     

Interaction of fatty acids with recombinant rat intestinal and liver fatty acid-binding proteins

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.     

Preparation of N-acylated proteins modified with fatty acids having a specific chain length using an insect cell-free protein synthesis system

To establish a strategy to generate N-acylated proteins modified with fatty acids having a specific chain length, tGelsolin-streptag, an epitope-tagged model protein having an N-myristoylation motif, was synthesized using an insect cell-free protein synthesis system in the presence of acyl-CoA with various fatty acid chain lengths. It was found that the fatty acid species attached to the N-termini fully depended on the acyl-CoA species added to the reaction mixture. N-Acylated proteins with fatty acid chain lengths of 8, 10, 12, and 14 were generated successfully.     

Quantitative aspects of free fatty acid metabolism in the fasted rat

Palmitate-1-(14)C was injected intravenously into unanesthetized, fasted rats. Disappearance of tracer from plasma free fatty acids was studied. A large component of free fatty acid (FFA) recycling was directly demonstrated by reinjection experiments. The latter studies also indicated the existence of an unidentified, rapidly turning over polar lipid in plasma which was synthesized from palmitate-(14)C. The appearance of (14)C in hepatic and extrahepatic triglycerides, in other esters, and in respired CO(2) was also followed. The data were analyzed using a multicompartmental model and a digital computer. Only a small fraction of the triglycerides formed in liver was derived directly from plasma free fatty acids. The major portion of net triglyceride formation appeared to be by way of an intermediate nontriglyceride ester pool which turned over relatively slowly compared to plasma free fatty acids. Initial approximations are as follows ( micromoles of fatty acid per min per 100 g body weight): net free fatty acid mobilization (irreversible disposal) = 2.4; hepatic triglyceride formation directly from plasma free fatty acid = 0.1; total hepatic lipid formation from plasma free fatty acids = 0.5; oxidation of free fatty acids to CO(2) = 0.8; percentage of respired CO(2) from direct oxidation of fatty acids = 12%; extrahepatic triglyceride formation directly from fatty acids = 0.4; total extrahepatic lipid formed directly from fatty acids = 1.2.     

Mapping of QTLs for oil content and fatty acid composition in Indian mustard [Brassica juncea (L.) Czern. and Coss.]

An AFLP linkage map of Brassica juncea (L.) Czern and Coss was constructed using 88 recombinant inbred lines (RILs) from a cross between an Indian cultivar ‘Varuna’ and an accession from Poland ‘BEC-144’. The map included 91 AFLP markers organized on 19 linkage groups covering a total map distance of 1679.1 cM. A total of 14 QTLs were detected for oil content (2 QTLs), erucic acid (2 QTLs), eicosenoic acid (2 QTLs), linolenic acid (3 QTLs), linoleic acid (3 QTLs) and palmitic acid (2 QTLs). A specific genomic region on LG2 was associated with contents of three fatty acids: erucic acid, eicosenoic acid and linoleic acid. Some of the markers showed absolute linkage with the QTLs associated with the levels of linolenic acid, linoleic acid and oil content. These markers may be used for improvement of fatty acid profile of B. juncea.     

Functional analysis of a beta-ketoacyl-CoA synthase gene,MpFAE2, by gene silencing in the liverwort Marchantia polymorpha L

We have isolated a beta-ketoacyl CoA synthase (KCS) gene, MpFAE2, from a liverwort, Marchantia polymorpha, and identified its substrate specificity using the technique of dsRNA-mediated gene silencing and overexpression. KCS catalyzes an essential reaction in the fatty acid elongation process, i.e., condensation of malonyl-CoA with acyl-CoA. By introducing a construct with a hairpin structure containing a partial MpFAE2 gene, the level of the MpFAE2 gene expression was suppressed constitutively. The transgenic plants showed a specific accumulation of fatty acid 18:0. In contrast, in transgenic M. polymorpha plants overexpressing the MpFAE2 gene, fatty acid 22:0 is accumulated. These results indicate that the MpFAE2 gene product catalyzes the elongation steps of 18:0 to 20:0 and possibly also of 20:0 to 22:0.