Effect of fatty acids of ω6 series on the biosynthesis of arachidonic acid in HTC cells

Summary The influence of the preincubation of HTC cells with fatty acids of 6 series and columbinic acid (St, 9c, 12c 18:3) on the biosynthesis of arachidonic acid was studied. The cells were incubated on a chemically defined medium with or without the addition of unlabeled linoleic, -linolenic, eicosatrienoic, arachidonic, docosatetraenoic, docosapentaenoic and columbinic acids. After 24 hr of preincubation in the presence of the aforementioned fatty acids, [1-¹⁴C]eicosa-8,11,14-trienoic acid was added to the culture medium as the only lipidic source. Twenty-four hours later the synthesis of arachidonic acid and the fatty acid composition of the cells were determined. At 20 MM concentration the 6 fatty acids studied except docosapentaenoic acid produced an increase on the biosynthesis of arachidonic acid compared to the cells incubated in the absence of unlabeled fatty acids in the medium. The fatty acids added to the culture medium were incorporated into the cells and modified their fatty acid composition. Columbinic acid, with a similar structure to linoleic acid, also produced a significant increase on the conversion of eicosatrienoic acid to arachidonic acid. These results would suggest that the effect of both, linoleic and columbinic acids, may be adscribed to their configuration and not necessarily to their transformation in higher homologs, since columbinic acid is unable to be desaturated.All authors are members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientifícas y Técnicas, Argentina.     

Repercussion of a deficiency in mitochondrial ß-oxidation on the carbon flux of short-chain fatty acids to the peroxisomal ß-oxidation cycle in Aspergillus nidulans

The fungus Aspergillus nidulans contains both a mitochondrial and peroxisomal ß-oxidation pathway. This work was aimed at studying the influence of mutations in the foxA gene, encoding a peroxisomal multifunctional protein, or in the scdA/echA genes, encoding a mitochondrial short-chain dehydrogenase and an enoyl-CoA hydratase, respectively, on the carbon flux to the peroxisomal ß-oxidation pathway. A. nidulans transformed with a peroxisomal polyhydroxyalkanoate (PHA) synthase produced PHA from the polymerization of 3-hydroxyacyl-CoA intermediates derived from the peroxisomal ß-oxidation of external fatty acids. PHA produced from erucic acid or heptadecanoic acid contained a broad spectrum of monomers, ranging from 5 to 14 carbons, revealing that the peroxisomal ß-oxidation cycle can handle both long and short-chain intermediates. While the ?foxA mutant grown on erucic acid or oleic acid synthesized 10-fold less PHA compared to wild type, the same mutant grown on octanoic acid or heptanoic acid produced 3- to 6-fold more PHA. Thus, while FoxA has an important contribution to the degradation of long-chain fatty acids, the flux of short-chain fatty acids to peroxisomal ß-oxidation is actually enhanced in its absence. While no change in PHA was observed in the ?scdA?echA mutant grown on erucic acid or oleic acid compared to wild type, there was a 2- to 4-fold increased synthesis of PHA in ?scdA?echA cells grown in octanoic acid or heptanoic acid. These results reveal that a compensatory mechanism exists in A. nidulans that increases the flux of short-chain fatty acids towards the peroxisomal ß-oxidation cycle when the mitochondrial ß-oxidation pathway is defective.     

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.     

Analysis of the β-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the β-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Δ³,Δ²-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10–25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through β-oxidation was more severely reduced in mutants devoid of Δ³,Δ²-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Δ³,Δ²-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.     

Construction of a series of intermediates in the β-oxidation pathway from THA to EPA via DHA in free acid form

β-Oxidation of most fatty acids occurs in the mitochondria. However, β-oxidation for ω-3 polyunsaturated fatty acids (PUFAs) is distinct from abundant fatty acids and occurs in the peroxisomes. Since little is known about peroxisomal β-oxidation, here we report the synthesis of proposed intermediates of ω-3 PUFA β-oxidation steps in free fatty acid form having a conjugated double bond, a β-hydroxyl group, a β-olefin and a β-carbonyl group. These fatty acids can serve as authentic samples for biological experiments.     

Seasonal variation in the lipid content of culturedLaminaria japonica: fatty acids,sterols, β-carotene and tocopherol

Seasonal variation in major lipid constituents of nutritional importance in culturedLaminaria japonica Aresch., such as fatty acids, sterols, -carotene and tocopherol, were investigated from December to October, the growing season. The total and saturated fatty acid contents were minimal in midsummer. Mono-unsaturated fatty acids gradually increased from late summer to autumn. The polyunsaturated fatty acid content (PUFA, (n-6) family) was maximal during warm months, while (n-3) PUFAs were most abundant during the cold months when algal thalli were very young, and decreased gradually toward October when sori had developed. Fucosterol content was maximum from February to June, but decreased steeply by nearly a half toward October, when 24-methylene cholesterol was highest although much less than fucosterol. The -carotene and tocopherol contents were maximal from July to September and slight during the winter.     

Effect of β-adrenergic agonist L-644,969 on the impact of the thermal environment on in vitro fatty acid synthesis and lipogenic enzymes in sheep

The effect of temperature and β-adrenergic agonist (BAA) on in vitro rates of fatty acid synthesis and catalytic activity of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) was examined in wether lambs after 5 weeks at either 0 or 20°C. Feeding BAA increased (P<0.05) rate of fatty acid synthesis by 38% in subcutaneous adipose (SC) tissue from cold-acclimated animals but the rate decreased (P<0.05) by 27% in SC tissue from warm-acclimated animals. In mesenteric fat (MS), BAA increased (P<0.05) fatty acid synthesis in the cold environment. In perirenal (PR) fat, rate of fatty acid synthesis was reduced (P<0.05) by 20% by BAA in the warm but had no effect in the cold. Activity of ACC in longissimus muscle was depressed (P<0.05) when BAA was fed in the warm environment. In adipose tissues BAA reduced (P<0.05) ACC activity in the warm, but reduced activity in the cold was limited to SC tissue. In PR tissue FAS activity was reduced (P<0.05) in the cold environment, while BAA increased FAS activity in the warm environment. Western blot analysis showed two isoforms of ACC with MW of 280 000 and 265 000 Da in longissimus muscle whereas only one isoform was recognized in each of Biceps femoris (280 000 Da) and adipose tissues (265 000 Da). Feeding BAA in the cold environment reduced (P<0.05) ACC and FAS immunoprotein expression in both MS and PR adipose tissues. The studies indicate that the effect of BAA on fatty acid synthesis and lipogenic enzymes is influenced by acclimation temperature.     

Effect of different carbon sources on the biosynthesis of polyunsaturated fatty acids ofα-linolenic acid family in culture of minimal deviation hepatoma 7288 c cells

Summary The effect of three different carbon sources on the biosynthesis of polyunsaturated fatty acids of the-linolenic acid series was investigated in hepatoma tissue culture (HTC) cells. Alpha linolenic acid was converted to higher homologs by a desaturating route that synthetized mainly 18:4 (6, 9, 12, 15), 20:4 (8, 11, 14, 17) and 20:5 (5, 8, 11, 14, 17) and an elongating route that produced 20:3 (11, 14, 17) and 20:4 (5, 11, 14, 17) acids. Fasting decreased both biosynthetic routes whereas glucose reactivated only the elongating pathway. Lactalbumin hydrolysate enhanced significantly only the desaturating route whereas glycerol was inactive. Glucose and aminoacids increased similarly the incorporation of labeled linolenic acid in the cells. The results are independent of hormonal effects.Members of the Carrera del Investigador Científico of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids

The protein encoded by the NCE103 gene of Candida glabrata, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K_As of 21.2–37 μM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.1–16.7 μM). The best activators were l-/d-Tyr, with activation constants of 7.1–9.5 μM. This study may bring a better understanding of the catalytic/activation mechanisms of β-CAs from pathogenic fungi.     

Salinomycin biosynthesis reversely regulates the β-oxidation pathway in Streptomyces albus by carrying a 3-hydroxyacyl-CoA dehydrogenase gene in its biosynthetic gene cluster

Streptomyces is well known for synthesis of many biologically active secondary metabolites, such as polyketides and non-ribosomal peptides. Understanding the coupling mechanisms of primary and secondary metabolism can help develop strategies to improve secondary metabolite production in Streptomyces. In this work, Streptomyces albus ZD11, an oil-preferring industrial Streptomyces strain, was proved to have a remarkable capability to generate abundant acyl-CoA precursors for salinomycin biosynthesis with the aid of its enhanced β-oxidation pathway. It was found that the salinomycin biosynthetic gene cluster contains a predicted 3-hydroxyacyl-CoA dehydrogenase (FadB3), which is the third enzyme of β-oxidation cycle. Deletion of fadB3 significantly reduced the production of salinomycin. A variety of experimental evidences showed that FadB3 was mainly involved in the β-oxidation pathway rather than ethylmalonyl-CoA biosynthesis and played a very important role in regulating the rate of β-oxidation in S. albus ZD11. Our findings elucidate an interesting coupling mechanism by which a PKS biosynthetic gene cluster could regulate the β-oxidation pathway by carrying β-oxidation genes, enabling Streptomyces to efficiently synthesize target polyketides and economically utilize environmental nutrients.     

CuI-promoted one-pot synthesis of N-Boc protected β-ketotriazole amino acids: application in the synthesis of new class of dipeptidomimetics

One-pot click chemistry of N(α)-Boc-bromomethylketones, NaN3 and propiolic acid affords N-Boc protected 1,4-disubstituted 1,2,3-β-ketotriazole acids in good to excellent yield. The use of CuI as catalyst and DMSO as solvent leads the click reaction to efficient, practical and column-free preparation of the title compounds. The utility of the resulting unnatural amino acids as building blocks to prepare triazole possessing peptidomimetics is also delineated.     

Effect of inhibitors of gibberellin biosynthesis on the induction of α-amylase in embryoless caryopses of Hordeum vulgare cv. Himalaya

The induction of -amylase by exogenously supplied gibberellin A₁ (GA₁) and GA₄ in embryoless caryopses of Hordeum vulgare (cv. Himalaya) was determined indirectly by measuring reducing sugars released from the endosperm. The presence of the inhibitors of GA biosynthesis, 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride (Amo 1618), Ancymidol, 2-chloroethyl trimethyl ammonium chloride (CCC) or (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl)pentan-3-ol (PP333) did not inhibit -amylase production by either GA₁ or GA₄.Abbreviations Amo-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidinecarboxylate methyl chloride - CCC 2-chloroethyl trimethyl ammonium chloride - cv. cultivar - GA gibberellin - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - PP333 (R,S)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,3-triazolyl) pentan-3-01     

Reconstruction of the astaxanthin biosynthesis pathway in rice endosperm reveals a metabolic bottleneck at the level of endogenous β-carotene hydroxylase activity

Astaxanthin is a high-value ketocarotenoid rarely found in plants. It is derived from β-carotene by the 3-hydroxylation and 4-ketolation of both ionone end groups, in reactions catalyzed by β-carotene hydroxylase and β-carotene ketolase, respectively. We investigated the feasibility of introducing an extended carotenoid biosynthesis pathway into rice endosperm to achieve the production of astaxanthin. This allowed us to identify potential metabolic bottlenecks that have thus far prevented the accumulation of this valuable compound in storage tissues such as cereal grains. Rice endosperm does not usually accumulate carotenoids because phytoene synthase, the enzyme responsible for the first committed step in the pathway, is not present in this tissue. We therefore expressed maize phytoene synthase 1 (ZmPSY1), Pantoea ananatis phytoene desaturase (PaCRTI) and a synthetic Chlamydomonas reinhardtii β-carotene ketolase (sCrBKT) in transgenic rice plants under the control of endosperm-specific promoters. The resulting grains predominantly accumulated the diketocarotenoids canthaxanthin, adonirubin and astaxanthin as well as low levels of monoketocarotenoids. The predominance of canthaxanthin and adonirubin indicated the presence of a hydroxylation bottleneck in the ketocarotenoid pathway. This final rate-limiting step must therefore be overcome to maximize the accumulation of astaxanthin, the end product of the pathway.     

Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?

Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n?=?10) or food deprived for 18?h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n?=?10), L: -leucine (1.35?g/kg, LEU, n?=?10), L: -citrulline (1.80?g/kg CIT, n?=?10) or isonitrogenous non-essential amino acids (NEAA, n?=?10). After gavage, the rats were injected with a flooding dose of [(13)C] valine to determine MPS. The rats were killed 50?min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P??CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.     

The plastidic 3-phosphoglycerate → acetyl-CoA pathway in barley leaves and its involvement in the synthesis of amino acids,plastidic isoprenoids and fatty acids during chloroplast development

Earlier studies on the synthesis of C₃-derived amino acids, plastidic isoprenoids and fatty acids from CO₂ by isolated chloroplasts in the light indicate the presence of a complete, but low-capacity, chloroplast (chlp) 3-phosphoglycerate acetyl-CoA pathway which is predominantely active in immature (developing) chloroplasts (A. Heintze et al., 1990, Plant Physiol. 93, 1121–1127). In this paper, we demonstrate the activity of the enzymes involved i.e. chlp phosphoglycerate mutase, chlp enolase, chlp pyruvate kinase and chlp pyruvate-dehydrogenase complex (PDC), in the stroma of purified barley (Hordeum sativum L.) chloroplasts of different developmental stages. The chlp phosphoglycerate mutase was partially purified for the first time. The activities of the enzymes of this chlp pathway (except PDC) were about a magnitude lower than those of the cytosolic enzymes. The chlp PDC of barley was more active than that of spinach. The apparent K _m values of the enzymes of this pathway were about 100 M or lower except for the chlp phosphoglycerate mutase which had a K _m of 1.6–1.8 mM for 3-phospho-d-glycerate. Interestingly, no appreciable change in the activity of these enzymes was observed during maturation of the chloroplasts. In contrast, the activity of the reversible NADP⁺-glyceraldehyde 3-phosphate dehydrogenase increased about five times (from 140 to 590 nkat per g leaf dry weight). The following hypothesis is put forward to explain the regulation of carbon metabolism during chloroplast development: 3-phospho-d-glycerate is withdrawn from a common pool by the actions of 3-phosphoglycerate kinase and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase, the activity of which increases considerably during maturation of chloroplasts. This leads to an insufficient supply of 3-phospho-glycerate for the chlp phosphoglycerate mutase, which has a low affinity for its substrate.Abbreviations C₃ C₂₅ pathway 3-phospho-d-glycerate acetyl-CoA pathway - Chl chlorophyll - chlp chloroplast(ic) - GAP d-glyceraldehyde-3-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - PDC pyruvate dehydrogenase complex - PEP phosphoenolpyruvate - 2- and 3-PGA 2- and 3-phospho-d-glycerate - U unit - mmol·min^t-1 (=16.67 nkat) This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG and Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie e. V., Frankfurt/Main, FRG, (scholarship to P.H.). The authors thank Dr. K.P. Heise (Institut für Biochemie der Pflanzen, Universität Göttingen, FRG) for the gas-liquid chromatography measurements, Gabriele Böl, Dietmar Budde, Daniel Gruber, Andreas Haaf, and Antje Wassmann (all Zentrum Biochemie, Medizinische Hochschule Hannover, FRG) and Kerstin Meereis, Martin Preiss, Uwe Schwanke (all Botanisches Institut, Tierärztliche Hochschule Hannover, FRG) for detailed and skillful work, Dr. Indra Willms-Hoff, Carola Leuschner and Dr. Christian L. Schmidt for constructive criticism, and Mrs. Saime Aydogdu for technical assistance.     

Effect of theβ-adrenoceptor agonist BRL 26830 on fatty acid synthesis and on the activities of pyruvate dehydrogenase and acetyl-CoA carboxylase in adipose tissues of the rat

BRL 26830 is a thermogenic-adrenoceptor agonist which stimulates lipolysis and fatty acid oxidationin vivo. It also stimulates insulin secretion, and hence promotes glucose utilisationin vivo. The effect of this agent on white and brown adipose tissue of the rat was investigated. BRL 26830 increased the rate of fatty acid synthesisin vivo in white adipose tissue by 135% but reduced the rate of fatty acid synthesisin vivo in brown adipose tissue by 78%. The increase was abolished in white adipose tissue of streptozotocin-diabetic rats, indicating that the effect involved a rise in circulating insulin levels. The reduction in fatty acid synthesis in brown adipose tissues was associated with a reduction in the activity of acetyl-CoA carboxylase in the tissue consistent with a direct-adrenoceptor-mediated effect. BRL 26830 also increased the proportion of pyruvate dehydrogenase in its active formin vivo in brown adipose tissue and this increase was abolished in streptozotocin-diabetic rats. These findings illustrate different sensitivities of white and brown adipose tissues to combined-adrenergic and insulin stimulation.