Determination of the secondary structural elements of chicken liver fatty acid binding protein by two‐dimensional homonuclear NMR

A conformational study in solution of the fatty acid binding protein from chicken liver is presented. The nearly complete sequence‐specific ¹H resonance assignment was achieved from homonuclear two‐dimensional nmr experiments using a sample of native protein. The principal elements of secondary structure were identified: 10 antiparallel β‐strands and one helical segment followed by a turn comprising 5 residues. These elements correspond closely with those of the crystal structure of the related protein, and two new secondary structural features obtained from the nmr data are the β‐sheet conformation between the first and the last β‐strand in the protein sequence, as well as a helical loop at the N‐terminus of the polypeptide chain. © 1999 John Wiley & Sons, Inc. Biopoly 50: 1–11, 1999     

肝型脂肪酸结合蛋白研究进展

肝型脂肪酸结合蛋白（liver fatty acid binding protein,L-FABP）是脂肪酸结合蛋白（fatty acid binding proteins,FABPs）家族重要的成员,在肝脏、小肠、肾脏等组织中均有表达。L-FABP在不饱和脂肪酸、饱和脂肪酸、胆固醇、胆汁酸等转运过程中扮演重要角色。目前研究显示L-FABP在脂肪肝、肝硬化以及肝癌发生发展中起到重要作用,并有望作为肝损伤的早期检测指标。此外,新近研究发现尿中L-FABP水平还可以用于预测1型糖尿病患者的临床结局。在2型糖尿病中,尿中L-FABP与糖尿病性肾病的病程有密切关系。主要就L-FABP的特性、结构及其与疾病的关系做一综述。     

The effect of glucagon-induced adenosine 3' ,5' -monophosphate concentrations on bile acid synthesis in isolated rat liver cells

The effect of increased intracellular adenosine 3' ,5' -monophosphate (cAMP) concentrations on bile acid synthesis in isolated rat hepatocytes was investigated. When the cells were incubated in the presence of glucagon (0.2 microM) and theophylline (1 mM) the observed rise in the level of cAMP was accompanied by an increase in bile acid production. Hepatocyte cAMP concentrations after 1 h of incubation showed a highly significant positive linear correlation with the amounts of bile acid synthesised by the cells during this time. These results suggest that bile acid production is related to the concentration of cAMP in isolated hepatocytes and provide evidence for a role for the cyclic nucleotide in the regulation of bile acid synthesis.     

Efficiency of fatty acid synthesis by oleaginous yeasts: Prediction of yield and fatty acid cell content from consumed C/N ratio by a simple method

In nitrogen-limited media, growth and fatty acid formation by the oleaginous yeast Rhodotorula glutinis, i.e., yield and fatty acid cell content, have been characterized regarding carbon and nitrogen availabilities. It was shown that the formation of fatty acid free biomass was limited by nitrogen availability, whereas the fatty acid production was directly dependent on the consumed C/N ratio. According to these observations, the fraction of substrate consumed for fatty acid synthesis was estimated by using a simple method based on the actual yields, i.e., the mass of carbon source strictly converted into fatty acids and fatty acid free biomass. From these results, relationships were established allowing to predict in a simple and performing manner the maximal attainable fatty acid cell content and yield from the available carbon and nitrogen. These relationships were validated by using experimental data obtained by various authors with different yeast strains, and the proposed method was compared to the energetic and mass balance method previously described.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Evidence for the synthesis of the multi-positional isomers of monounsaturated fatty acid in Methylococcus capsusatus by the anaerobic pathway

Abstract The biosynthesis of the positional isomers of the monounsaturated fatty acids of Methylococcus capsulatus (Bath) has been investigated by studying the incorporation of [2-¹⁴C]malonyl CoA into long-chain fatty acids in vitro. The major unsaturated products were Δ ⁹ 16:1 and Δ ¹¹ 18:1; however, Δ ⁸, Δ ¹⁰ and Δ ¹¹ 16:1, as well as, Δ ¹⁰, Δ ¹² and Δ ¹³ 18:1 were also synthesized. The exclusion of O₂ from the reaction vessel did not affect the synthesis of unsaturated fatty acids or the double bonds positions. Cerulenin inhibited the synthesis of unsaturated fatty acid more than saturated fatty acid. The use of both [1-¹⁴C] octanoate and [1-¹⁴C] decanoate as substrate resulted in the synthesis of long-chain fatty acids, however, unsaturates were only synthesized from octanoate. These results imply that the unique positional isomers of M. capsulatus are not synthesized by an aerobic mechanism.     

Effects of osmolytes on unfolding of chicken liver fatty acid synthase

Urea-induced aggregation of chicken liver fatty acid synthase [acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating,oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85 ] was studied. The aggregation was facilitated at increased ionic strength. Methyl--cyclodextrin and some osmolytes, such as glycerol, sucrose, proline, glycine, and heparin, could effectively prevent the aggregation, implying an artificial chaperone role of those substances during fatty acid synthase unfolding. The osmolytes also protected the enzyme from inactivation.     

Effect of thiocarbamate herbicides on fatty acid synthesis by potato

S-ethyldipropylthiocarbamate (EPTC), S-(2,3-dichloroallyl)diisopropylthiocarbamate (diallate) and S-(2,3,3-trichloroallyl)diisopropylthiocarbamate (triallate) inhibited the formation of very long chain fatty acids by aged potato discs. Incorporation of acetate-[¹⁴C] into total fatty acids was inhibited 24% by EPTC, 50% by triallate and 55% by diallate at 10^?4 M. The relative sensitivity of very long chain fatty acid synthesis to thiocarbamates in potato tuber provides further evidence that these herbicides reduce cuticular wax by inhibiting fatty acid elongation.     

Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica

The effect of solvents and solvent mixtures on the synthesis of myristic acid esters of different carbohydrates with an immobilized lipase from C. antarctica was investigated. The rate of myristyl glucose synthesized by the enzyme was increased from 3.7 to 20.2 micromol min(-1) g(-1) by changing the solvent from pure tert-butanol to a mixture of tert-butanol:pyridine (55:45 v/v), by increasing the temperature from 45 degrees C to 60 degrees C, and by optimizing the relative amounts of glucose, myristic acid, and the enzyme preparation. Addition of more than 2% DMSO to the tert-butanol:pyridine system resulted in a reduction of enzyme activity. Lowering the water content of the enzyme preparation below 0.85% (w/w) resulted in significant decreases in enzyme activity, while increasing the water content up to 2.17% (w/w) did not significantly affect the enzyme activity. The highest yields of myristyl glucose were obtained when an excess of unsolubilized glucose was present in the reaction system. In this case, all of the initially solubilized and a significant amount of the initially unsolubilized glucose was converted to the ester within 24 h of incubation, resulting in a myristyl glucose concentration of 34 mg/mL(-1). Myristic acid esters of fructose (22.3 micromol min(-1) g(-1)), alpha-D-methyl-glucopyranoside (26.9 micromol min(-1) g(-1)) and maltose (1.9 micromol min(-1) g(-1)) could also be prepared using the tert-butanol:pyridine solvent system. No synthesis activity was observed with maltotriose, cellobiose, sucrose, and lactose as substrate.     

L-acetylcarnitine, a substrate for chloroplast fatty acid synthesis

Abstract. H¹⁴CO₃ was not incorporated into fatty acids by isolated pea leaf chloroplasts, which, therefore, do not possess a self-contained pathway for the synthesis of fatty acids from early intermediates of the Calvin cycle. Citrate, pyruvate, acetate and L-acetylcarnitine were all shown to act as sources of acetyl groups for fatty acid synthesis by pea leaf chloroplasts. L-acetylcarnitine was the best substrate, being incorporated into fatty acids at rates that were at least five-fold higher than those achieved with the other substrates. Citrate was incorporated into fatty acids at the lowest rate, followed by pyruvate, with acetate being incorporated at the second highest rate of all. When the isolated chloroplasts were ruptured, an inhibition of L-acetylcarnitine incorporation into fatty acids was noted, whilst acetate incorporation remained unaffected. L-acetylcarnitine also increased the ratio of monoenoic: saturated fatty acids synthesized, compared with a 1:1 ratio observed when citrate, pyruvate and acetate were supplied as substrates. It is suggested that L-carnitine and carnitine acyltransferases play a central role in plant acyl CoA metabolism by facilitating the transfer of activated acyl groups across membranes (acyl CoA barriers).     

Diltiazem inhibits fatty acid oxidation in the isolated perfused rat liver

The effects of diltiazem on fatty acid metabolism were measured in the isolated perfused rat liver and in isolated mitochondria. In the perfused rat liver diltiazem inhibited oxygen uptake and ketogenesis from endogenous substrates. Ketogenesis from exogenously supplied palmitate was also inhibited. The β-hydroxybutyrate/acetoacetate ratio in the presence of palmitate alone was equal to 3·2. When the fatty acid and diltiazem were present simultaneously this ratio was decreased to 0·93, suggesting that, in spite of the inhibition of oxygen uptake, the respiratory chain was not rate limiting for the oxidation of the reducing equivalents coming from β-oxidation. In experiments with isolated mitochondria, incubated in the presence of all intermediates of the Krebs cycle, pyruvate or glutamate, no significant inhibition of oxygen uptake by diltiazem was detected. Inhibition of oxygen uptake in isolated mitochondria was found only when palmitoyl CoA was the source of the reducing equivalents. It was concluded that a direct effect on β-oxidation may be a major cause for the inhibition of oxygen uptake caused by diltiazem in the perfused liver. © 1997 John Wiley & Sons, Ltd.     

Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids

Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.     

Synthesis of esters of tetradecanoic acid deuterated at the penultimate carbon: Some general procedures for the synthesis of selectively deuterated fatty acids

Methyl tetradecanoate, deuterated in the penultimate position has been prepared by three synthetic routes. One method in which an oxoester is an intermediate, utilizes the selective reducing properties of sodium borodeuteride and sodium cyanoborodeuteride towards oxo, tosyloxy and ester groups to introduce the deuterium label at only the oxo carbon of the oxoester. The second procedure, introduces a single deuteriumat at the oxo carbon of an oxoester, in a selective deoxygenation step which reduces an intermediate tosylhydrazone with sodium cyanoborodeuteride in acidic dimethylformamide-sulfolane. The third pathway, couples lithium dimethylcuprate (I) to the tosylate derivative of a ω-deuterio-ω-hydroxy methylester, prepared from the half-ester of a dicarboxylic acid. Utilizing suitable modifications, these three methods should be generally applicable in the synthesis of any saturated straight-chain alkanoate, selectively deuterated in the penultimate (ω − 1) carbon, or at any other carbon in the hydrocarbon chain.     

Metabolic profiles of fish nodavirus infection in vitro: RGNNV induced and exploited cellular fatty acid synthesis for virus infection

Red‐spotted grouper nervous necrosis virus (RGNNV), the causative agent of viral nervous necrosis disease, has caused high mortality and heavy economic losses in marine aquaculture worldwide. However, changes in host cell metabolism during RGNNV infection remain largely unknown. Here, the global metabolic profiling during RGNNV infection and the roles of cellular fatty acid synthesis in RGNNV infection were investigated. As the infection progressed, 71 intracellular metabolites were significantly altered in RGNNV‐infected cells compared with mock‐infected cells. The levels of metabolites involved in amino acid biosynthesis and metabolism were significantly decreased, whereas those that correlated with fatty acid synthesis were significantly up‐regulated during RGNNV infection. Among them, tryptophan and oleic acid were assessed as the most crucial biomarkers for RGNNV infection. In addition, RGNNV infection induced the formation of lipid droplets and re‐localization of fatty acid synthase (FASN), indicating that RGNNV induced and required lipogenesis for viral infection. The exogenous addition of palmitic acid (PA) enhanced RGNNV infection, and the inhibition of FASN and acetyl‐CoA carboxylase (ACC) significantly decreased RGNNV replication. Additionally, not only inhibition of palmitoylation and phospholipid synthesis, but also destruction of fatty acid β‐oxidation significantly decreased viral replication. These data suggest that cellular fatty acid synthesis and mitochondrial β‐oxidation are essential for RGNNV to complete the viral life cycle. Thus, it has been demonstrated for the first time that RGNNV infection in vitro overtook host cell metabolism and, in that process, cellular fatty acid synthesis was an essential component for RGNNV replication.     

Effects of zinc deficiency on the fatty acid composition and metabolism in rats fed a fat-free diet

The effects of dietary zinc deficiency (ZD) on the composition and metabolism of the fatty acyl chains of phospholipids in rat liver were investigated with a fat-free diet. The levels of (n−9) fatty acids such as 18∶1 and 20∶3(n−9) in liver phospholipids (PL) were significantly lower in ZD-rats (19.4% and 5.4%, respectively) than in PF-rats (25.2 and 8.3%). On the other hand, the level of (n−6) acids such as 18∶2 and 20∶4 were higher in ZD-rats (3.3 and 19.1%, respectively) than in PF-rats (2.1 and 14.9%). In order to study the metabolism of fatty acids in vivo,¹⁴C-18∶0 or¹⁴C-18∶2 was intravenously injected, and then the conversion to the respective metabolite was examined. After the injection of¹⁴C-18∶0, the radioactivity was found in 18∶0 (49.3% of the total), 18∶1 (33.2%), and 20∶3 (n−9) (9.1%) in liver PL in PF-rats at 24h. In ZD-rats, the radioactivity was dramatically lower in 18∶1 (23.5%) and 20∶ (n−9) (3.6%), suggesting that the conversion of 18∶0 to 18∶1 and 20∶3 (n−9) was strongly inhibited in ZD-rats. When¹⁴C-18∶2 was injected, the radioactivity was mainly found in 18∶2, 20∶3(n−6), and 20∶4. The radioactivity in 20∶4 in ZD-rats was slightly higher than that in control rats. These results indicate that zinc deficiency affects the fatty acid metabolism in liver, in particular, it causes a reduction in δ9 desaturase activity, when rats are fed a fat-free diet.     

In vitro desaturation or elongation of monotrans isomers of linoleic acid by rat liver microsomes

Several nutritional studies have shown the in vivo conversion of the 9c,12t-18:2 and 9t,12c-18:2 into long chain polyunsaturated fatty acids (PUFA) containing 20 carbons (geometrical isomers of eicosadienoic and eicosatetraenoic acids). In the present work, some in vitro studies were carried out in order to have precise information on the conversion of these two isomers.In a first set of experiments, studies were focused on the in vitro 6 desaturation, the first regulatory step of the biosynthesis of n-6 long chain PUFA, from 9c,12c-18:2. Rat liver microsomes were prepared and incubated under desaturation conditions with [1-¹⁴C]-9c,12c-18:2 in presence of unlabelled 9c,12t-, 9t,12c- or 9t,12t-18:2. The data show that each trans isomer induced a decrease of the 6 desaturation of the [1-¹⁴C]-9c,12c-18:2, but the 9c,12t-18:2 was the most potent inhibitor (up to 63%). Rat liver microsomes were also incubated with [1-¹⁴C]-9c,12c-18:2, [1-¹⁴C]-9c,12t-18:2 or [1-¹⁴C]-9t,12c-18:2 under desaturation conditions. The results indicated that 18:2 9c,12t is a much better substrate for desaturase than 9t,12c-18:2. Moreover, the conversion levels of [1-¹⁴C]-9c,12t-18:2 was similar to what was observed for its all cis homologue, at low substrate concentration only. In a second set of experiments, in vitro elongation studies of each mono-trans 18:2 isomers and 9c,12c-18:2 were carried out. For that purpose, rat liver microsomes were incubated with [1-¹⁴C]-9c,12c-18:2, [1-¹⁴C]-9c,12t-18:2 or [1-¹⁴C]-9t,12c-18:2 under elongation conditions. The data show that [1-¹⁴C]-9t,12c-18:2 is better elongated than 9c,12c-18:2 while the amount of product formed from [1-¹⁴C]-9c,12t-18:2 was lower than was produced from the 9c,12c-18:2.Thus, the desaturation enzymes presented a higher affinity for the 9c,12t-18:2 whereas the elongation enzyme presented a higher affinity for the 9t,12c-18:2.     

One-pot synthesis of polyunsaturated fatty acid amides with anti-proliferative properties

A one-pot environmentally friendly transamidation of ω-3 fatty acid ethyl esters to amides and mono- or diacylglycerols was investigated via the use of a polymer-supported lipase. The method was used to synthesize a library of fatty acid monoglyceryl esters and amides. These new derivatives were found to have potent growth inhibition effects against A549 lung cancer cells.