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.     

Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Kinetic advantage of the interaction between the fatty acid β-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C₄, C₈ and C₁₄)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD⁺ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 μM in gently sonicated and 15 μM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD⁺-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD⁺ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of β-oxidation enzyme in situ. Respiration-linked oxidation of bytyryl-, oxtanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

Effect of γ irradiation on the fatty acid composition of soybean and soybean oil

Food irradiation is a form of food processing to extend the shelf life and reduce spoilage of food. We examined the effects of γ radiation on the fatty acid composition, lipid peroxidation level, and antioxidative activity of soybean and soybean oil which both contain a large amount of unsaturated fatty acids. Irradiation at 10 to 80 kGy under aerobic conditions did not markedly change the fatty acid composition of soybean. While 10-kGy irradiation did not markedly affect the fatty acid composition of soybean oil under either aerobic or anaerobic conditions, 40-kGy irradiation considerably altered the fatty acid composition of soybean oil under aerobic conditions, but not under anaerobic conditions. Moreover, 40-kGy irradiation produced a significant amount of trans fatty acids under aerobic conditions, but not under anaerobic conditions. Irradiating soybean oil induced lipid peroxidation and reduced the radical scavenging activity under aerobic conditions, but had no effect under anaerobic conditions. These results indicate that the fatty acid composition of soybean was not markedly affected by radiation at 10 kGy, and that anaerobic conditions reduced the degradation of soybean oil that occurred with high doses of γ radiation.     

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.     

1-Butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid β-oxidation

Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid β-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions of the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde dehydrogenase AdhE from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of reversed action of fatty acid β-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.     

Modulation of lipogenic enzymes, fatty acid synthase and Δ9-desaturase, in relation to migration in the western sandpiper (Calidris mauri)

Long-distance migration in birds is characterized physiologically by periods of rapid fattening and lipogenesis, and increased desaturation of fatty acids stored in adipose tissue. We investigated seasonal, age- and sex-related differences in activities of two lipogenic enzymes, fatty acid synthase and Δ⁹-desaturase, in relation to migration in the small, Arctic-nesting western sandpiper (Calidris mauri). Migration, and associated lipogenesis and fattening, involved marked upregulation of these enzymes in this species. However, this increase in enzyme activity was only seen in actively migrating birds during spring migration, when fatty acid synthase and Δ⁹-desaturase levels increased by 53% and 113%, respectively, compared to non-migrating birds. There was no change in fatty acid synthase enzyme activity during the premigration period, even though body mass of adult birds increased significantly during this period. Similarly, there was no increase in Δ⁹-desaturase activity during premigration, despite the fact that birds increase the proportion of monounsaturated fatty acids in their fat stores at this time. We suggest that upregulation of lipogenic enzymes is required to support high rates of mass gain (0.4 g day⁻¹) during short (1–4 day) periods at stop-over sites. However, slower rates of mass gain (0.09 g day⁻¹) over several weeks prior to migration can be achieved without any increase in tissue-specific enzyme activity. Accepted: 29 November 1999     

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.     

Metabolomics reveals the mechanism of (−)-hydroxycitric acid promotion of protein synthesis and inhibition of fatty acid synthesis in broiler chickens

(−)-Hydroxycitric acid (HCA), a major component of Garcinia cambogia extracts, has been shown to suppress BW gain and fat accumulation in animals and humans. However, the mechanism remains unknown. In this study, gas chromatography-mass spectrometry was used to analyse serum metabolites, and principal component analysis and partial least-squares-discriminant analysis models were generated to analyse serum metabolite changes in broiler chickens after the administration of (−)-HCA at 0, 1000, 2000 and 3000 mg/kg diets for 28 days. Metabolites showing significant changes were screened by ‘variable importance in the projection’ plots. The results showed that 20 metabolites in the 1000 mg/kg (−)-HCA treatment group and 16 metabolites in 3000 mg/kg (−)-HCA treatment group were significantly altered. Metabolites pathway enrichment analysis indicated that these metabolites were mainly associated with metabolism of amino acids, protein synthesis, citric acid cycle, and uric acid and fatty acid synthesis. The data indicated that (−)-HCA promoted protein synthesis by regulating the metabolic directions of amino acids. At the same time, (−)-HCA treatment inhibited fatty acid synthesis by promoting the citric acid cycle, resulting in reduced cytosolic acetyl-CoA content in broiler chickens. The present study identified global changes in metabolites and analysed the main canonical metabolic pathways in broiler chickens supplemented with (−)-HCA. These results will deepen our understanding of the mechanism of (−)-HCA’s effects in animals.     

Long time molecular dynamic simulation on the agonist binding and activation of the β2-adrenergic receptor

β₂-Adrenergic receptor (β₂AR) plays a critical role in mediating the effects of catecholamine hormones. Due to the flexibility of the structure of its active state, study of agonist–β2AR is usually performed by molecular dynamic (MD) simulation. In this study we show the representative characteristics of agonist binding and activation on β₂AR by MD simulation. The binding site and the conformational changes in the specific regions of β2AR are reasonable which confirmed the conclusion that agonist–β2AR reached its active-like state. We have studied the disruption of non-covalent intramolecular interactions, including the conserved DRY motif, the rotamer toggle switch and the ionic lock, the cytoplasmic ends of transmembranes 5 and 6, and some water-mediated hydrogen bond network regions. We conclude that agonist induced β₂AR to its active conformation, or at least the active-like conformation.     

Role of fatty acid uptake and fatty acid β-oxidation in mediating insulin resistance in heart and skeletal muscle

Fatty acids are a major fuel source used to sustain contractile function in heart and oxidative skeletal muscle. To meet the energy demands of these muscles, the uptake and β-oxidation of fatty acids must be coordinately regulated in order to ensure an adequate, but not excessive, supply for mitochondrial β-oxidation. However, imbalance between fatty acid uptake and β-oxidation has the potential to contribute to muscle insulin resistance. The action of insulin is initiated by binding to its receptor and activation of the intrinsic protein tyrosine kinase activity of the receptor, resulting in the initiation of an intracellular signaling cascade that eventually leads to insulin-mediated alterations in a number of cellular processes, including an increase in glucose transport. Accumulation of fatty acids and lipid metabolites (such as long chain acyl CoA, diacylglycerol, triacylglycerol, and/or ceramide) can lead to alterations in this insulin signaling pathway. An imbalance between fatty acid uptake and oxidation is believed to be responsible for this lipid accumulation, and is thought to be a major cause of insulin resistance in obesity and diabetes, due to lipid accumulation and inhibition of one or more steps in the insulin-signaling cascade. As a result, decreasing muscle fatty acid uptake can improve insulin sensitivity. However, the potential role of increasing fatty acid β-oxidation in the heart or skeletal muscle in order to prevent cytoplasmic lipid accumulation and decrease insulin resistance is controversial. While increased fatty acid β-oxidation may lower cytoplasmic lipid accumulation, increasing fatty acid β-oxidation can decrease muscle glucose metabolism, and incomplete fatty acid oxidation has the potential to also contribute to insulin resistance. In this review, we discuss the proposed mechanisms by which alterations in fatty acid uptake and oxidation contribute to insulin resistance, and how targeting fatty acid uptake and oxidation is a potential therapeutic approach to treat insulin resistance.     

Effect of ethionine on the synthesis of β-galactosidase inEscherichia coli

Ethionine at concentrations of 10⁻³M, 5×10⁻³M and 10⁻²M inhibits growth, both of β-galactosidase inducible ML-30 and constitutive ML-308Escherichia coli strains. The protein synthesis (measured by the incorporation of l-leucine-¹⁴C and l-aspartic-¹⁴C acid into proteins) of these strains is inhibited to the same extent as their growth. The synthesis of inducible and constitutive β-galactosidase produced by the strains ML-30 and ML-308, respectively, is considerably inhibited by ethionine.     

Activities of fatty acid desaturases and fatty acid composition of liver microsomes in rats fed β-carotene and 13-cis-retinoic acid

The fatty acid composition of microsomal lipids and the activities of Δ9- and Δ6-desaturases in liver microsomes of rats fed diets supplemented with β-carotene and two levels of 13-cis-retinoic acid were studied. Four groups of male, weanling rats were fed semipurified diets containing 0 or 100 mg β-carotene per kg diet, and 20 or 100 mg 13-cis-retinoic acid per kg diet. After 11 weeks of feeding, the rats were killed, liver microsomes were prepared and assayed for Δ9-desaturase and Δ6-desaturase activities. The activity of Δ9-desaturase was lower in liver microsomes of rats fed β-carotene-supplemented diet or the diet supplemented with the higher level of 13-cis-retinoic acid. Microsomal Δ6-desaturase activity was, however, higher in liver of rats fed 13-cis-retinoic acid; there was no effect of β-carotene on Δ6-desaturase activity. The fatty acid compositional data on total lipids of liver microsomes were consistent with the diet-induced changes in fatty acid desaturases. Phospholipid composition of liver microsomes was also altered as a result of feeding β-carotene or 13-cis-retinoic acid-containing diets. The proportions of phosphatidylethanolamine were generally higher, whereas those of phosphatidylcholine were lower in the experimental groups as compared with the control.     

Synthesis and in vitro evaluation of derivatives of the β₁-adrenergic receptor antagonist HX-CH 44

Isopropyl- and fluoroisopropyl-amino derivatives of the β(1)-adrenergic receptor antagonist 2-[4-[3-(tert-butyl-amino)-2-hydroxypropoxy]phenyl]-3-methyl-6-methoxy-4(3H)-quinazolinone ((±)HX-CH 44) were synthesized, including a concise and efficient preparation of the core, 2-(4-hydroxyphenyl)-6-methoxy-3-methylquinazolin-4(3H)-one. In vitro binding assays showed that the fluorinated analog was selective towards β(1)-adrenergic receptors over β(2)-adrenergic and 5-HT(1A) receptors. An X-ray crystallographic characterization of the fluorinated analog is also reported.     

The impact of fatty acids on the antibacterial properties of N-thiolated β-lactams

Bacterial fatty acid synthesis (FAS) is a potentially important, albeit controversial, target for antimicrobial therapy. Recent studies have suggested that the addition of exogenous fatty acids (FAs) to growth media can circumvent the effects of FAS-targeting compounds on bacterial growth. Consequently, such agents may have limited in vivo applicability for the treatment of human disease, as free FAs are abundant within the body. Our group has previously developed N-thiolated β-lactams and found they function by interfering with FAS in select pathogenic bacteria, including MRSA. To determine if the FAS targeting activity of N-thiolated β-lactams can be abrogated by exogenous fatty acids, we performed MIC determinations for MRSA strains cultured with the fatty acids oleic acid and Tween 80. We find that, whilst the activity of the known FAS inhibitor triclosan is severely compromised by the addition of both oleic acid and Tween 80, exogenous FAs do not mitigate the antibacterial activity of N-thiolated β-lactams towards MRSA. Consequently, we propose that N-thiolated β-lactams are unique amongst FAS-inhibiting antimicrobials, as their effects are unimpeded by exogenous FAs.     

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.     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane     

Effect of mutation Arg91Gly on the thermal stability of β-tropomyosin

The mutation Arg91Gly (R91G) in β-tropomyosin (β-TM) is known to cause distal arthrogryposis, a severe congenital disorder of muscle tissues. The influence of this mutation in β-TM on its structure and thermal denaturation was demonstrated. It was shown by the differential scanning calorimetry and circular dichroism that this point mutation dramatically decreased the thermal stability of the significant part of the β-TM (about a half of the molecule). This part of the β-TM molecule carrying R91G mutation unfolds at ～28°C, i.e., at a much lower temperature than the other part of the molecule, which melts at ～40°C. The data of the differential scanning calorimetry were compared with the results of temperature dependence of pyrene eximer fluorescence, which decreased upon the dissociation of two β-TM chains in the region of pyrene-labeled Cys-36. This comparison allowed one to conclude that this thermal transition reflected the thermal unfolding of the whole N-terminal part of β-TM. Interestingly, the destabilizing effect of Arg91Gly mutation spread for a rather long distance along the tropomyosin coiled-coil indicating a high cooperativity of the thermal denaturation within this part of β-TM.