Probing in vivo metabolism by stable isotope labeling of storage lipids and proteins in developing Brassica napus embryos

Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mM carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mM amino acids as well as 6 to 15 mM malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing (13)C-labeled carbohydrates. The (13)C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of (13)C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of (13)C label into plastid-formed fatty acids, but substantially diluted (13)C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. (13)C label in the terminal acetate units of C(20) and C(22) fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute (13)C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos.     

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.     

Fatty acid synthesis from amino acids in sheep adipose tissue

The rates of incorporation of 14C from 14C labelled acetate, glucose, alanine, leucine, isoleucine and valine into fatty acids has been measured in perirenal adipose tissue from foetal lambs and 8-month-old sheep, and into both fatty acids and acylglycerol glycerol in adipose tissue from 3-year-old sheep and 220-240 g female rats. Rates of incorporation of 14C from amino acids into fatty acids were much lower in adipose tissue from sheep (at all three ages) than from rats, whereas rates of incorporation of 14C into acylglycerol glycerol were either greater in sheep adipose tissue or the same as in rat adipose tissue. The rate of incorporation of 14C from amino acids into fatty acids decreased in the order leucine greater than alanine greater than isoleucine greater than valine in adipose tissue from rats and foetal lambs, and in the order leucine greater than alanine = isoleucine greater than valine in adipose tissue from 8-month- and 3-year-old sheep. Amino acids make a very small contribution to fatty acid synthesis in adipose tissue from sheep at all stages of development examined while fatty acids are a minor product of amino acid metabolism in sheep adipose tissue. The study provides further evidence for an important role for ATP-citrate lyase in restricting the utilization of acetyl-CoA generated in the mitochondria for fatty acid synthesis.     

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.     

A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic beta-cells

To gain insight into the relationship between acyl coenzyme A (CoA) esters and glucose-induced insulin release, acyl-CoA profiles were determined in clonal pancreatic beta-cells (HIT). A high sensitivity high performance liquid chromatography method was used to measure malonyl, succinyl, beta-hydroxy beta-methylglutaryl and acetyl-CoA esters and free CoASH. Malonyl-CoA content increased more than 3-fold following exposure of HIT cells to 10 mM glucose. The rise in malonyl-CoA, which preceded insulin secretion, was evident 2 min after exposure to glucose and was sustained for at least 30 min. The increase in malonyl-CoA was associated with inhibition of fatty acid oxidation, increased de novo lipid synthesis and a rise in diacylglycerol content. Succinyl-CoA levels, which may reflect anaplerotic influx into the citric acid cycle, were elevated in the presence of glucose. The concentration of acetyl-CoA and the ratio of free CoASH to acetyl-CoA was unchanged. The data are consistent with a metabolic model in which malonyl-CoA mediates the switch from fatty acid catabolism to lipid synthesis during glucose stimulation of beta-cells. We suggest that these changes in lipid metabolism, by leading to increased diacylglycerol synthesis or protein acylation could play a pivotal role in the regulation of the sustained phase of insulin secretion.     

Role of carnitine acetyltransferases in acetyl coenzyme A metabolism in Aspergillus nidulans

The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while β-oxidation of fatty acids generates acetyl-CoA in peroxisomes. The acetyl-carnitine shuttle in which acetyl-CoA is reversibly converted to acetyl-carnitine by carnitine acetyltransferase (CAT) enzymes is important for intracellular transport of acetyl units. In the filamentous ascomycete Aspergillus nidulans, a cytoplasmic CAT, encoded by facC, is essential for growth on sources of cytoplasmic acetyl-CoA while a second CAT, encoded by the acuJ gene, is essential for growth on fatty acids as well as acetate. We have shown that AcuJ contains an N-terminal mitochondrial targeting sequence and a C-terminal peroxisomal targeting sequence (PTS) and is localized to both peroxisomes and mitochondria, independent of the carbon source. Mislocalization of AcuJ to the cytoplasm does not result in loss of growth on acetate but prevents growth on fatty acids. Therefore, while mitochondrial AcuJ is essential for the transfer of acetyl units to mitochondria, peroxisomal localization is required only for transfer from peroxisomes to mitochondria. Peroxisomal AcuJ was not required for the import of acetyl-CoA into peroxisomes for conversion to malate by malate synthase (MLS), and export of acetyl-CoA from peroxisomes to the cytoplasm was found to be independent of FacC when MLS was mislocalized to the cytoplasm.     

The purification and function of acetyl coenzyme A:acyl carrier protein transacylase

When individual enzyme activities of the fatty acid synthetase (FAS) system were assayed in extracts from five different plant tissues, acetyl-CoA:acyl carrier protein (ACP) transacylase and beta-ketoacyl-ACP synthetases I and II had consistently low specific activities in comparison with the other enzymes of the system. However, two of these extracts synthesized significant levels of medium chain fatty acids (rather than C16 and C18 acid) from [14C]malonyl-CoA; these extracts had elevated levels of acetyl-CoA:ACP transacylase. To explore the role of the acetyl transacylase more carefully, this enzyme was purified some 180-fold from spinach leaf extracts. Varying concentrations of the transacylase were then added either to spinach leaf extracts or to a completely reconstituted FAS system consisting of highly purified enzymes. The results suggested that: (a) acetyl-CoA:ACP transacylase was the enzyme catalyzing the rate-limiting step in the plant FAS system; (b) increasing concentration of this enzyme markedly increased the levels of the medium chain fatty acids, whereas increase of the other enzymes of the FAS system led to increased levels of stearic acid synthesis; and (c) beta-ketoacyl-ACP synthetase I was not involved in the rate-limiting step. It is suggested that modulation of the activity of acetyl-CoA:ACP transacylase may have important implications in the type of fatty acid synthesized, as well as the amount of fatty acids formed.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.     

Lactate carbon does not enter the sugars of lipopolysaccharide when gonococci are grown in a medium containing glucose and lactate: implications in vivo

In media containing glucose, lactate stimulates the metabolism of gonococci at concentrations that simulate conditions in vivo. Nuclear magnetic resonance (NMR) spectroscopy of (13)C-labelled lipids obtained from gonococci grown in a synthetic medium with (13)C-labelled lactate and unlabelled glucose (culture A), (13)C-labelled glucose alone (culture B) or (13)C-labelled glucose and unlabelled lactate (culture C) showed lactate carbon was not present in glycerol/ethanolamine residues of lipids from culture A. This indicated that, in the presence of glucose, lactate gluconeogenesis is shut down. Hence, the stimulation of metabolism could result from the production of extra energy because lactate is used solely for conversion to acetyl-CoA, the precursor of fatty acid synthesis and the components of the tricarboxylic acid cycle. In this paper, additional evidence for lack of gluconeogenesis has been sought using a different approach. The carbohydrate moieties of lipopolysaccharide (LPS) have been examined for lactate carbon after gonococci were grown with lactate and glucose. Two methods were used: NMR spectroscopy of (13)C-labelled lipopolysaccharide purified from the three cultures described above showed that, in the presence of glucose, lactate carbon, in contrast to glucose carbon, was not in the carbohydrate moiety. Also, (14)C-labelled lactate was added to a culture containing unlabelled glucose and lactate (culture A) and [(14)C]glucose to cultures containing unlabelled glucose without unlabelled lactate (culture B) and with unlabelled lactate (culture C). When LPS samples purified from these cultures were subjected to hydrazinolysis, the ratio of the radioactivity of water-soluble products (carbohydrate moieties) to those of chloroform-soluble products (fatty acids) was much lower when [(14)C]lactate was used in culture A, than when [(14)C]glucose was used in cultures B and C. Thus, in the presence of glucose, lactate carbon, unlike glucose carbon, is incorporated predominantly into fatty acids of LPS, not into its carbohydrate moieties. There is no doubt, therefore, that gluconeogenesis is shut off when lactate is present with glucose and there is a consequent stimulation of metabolism. This probably occurs in vivo on mucous surfaces, where gonococci are surrounded by a mixture of glucose and lactate in the secretions.     

The physiological role of oxygen-sensitive pyruvate dehydrogenase in mitochondrial fatty acid synthesis in Euglena gracilis

In Euglena gracilis a malonyl-CoA-independent fatty acid-synthetic system, in which fatty acids are synthesized directly from acetyl-CoA as both primer and C2 donor, occurs in mitochondria, and the system contributes to the wax ester fermentation. The activity of fatty acid synthesis in the mitochondrial system was enhanced about six times when an artificial acetyl-CoA-regenerating system was present, indicating that the fatty acid-synthetic activity is controlled by the ratio of acetyl-CoA against CoA. When fatty acids were synthesized using pyruvate instead of acetyl-CoA as substrate, a high activity, about 30 times higher than that from acetyl-CoA, was found under anaerobic conditions (below 10(-5) M oxygen), while in aerobiosis fatty acids were not synthesized at all. CoA, NADH, and NADP+ were required as cofactors for fatty acid synthesis from pyruvate. It was indicated that high activity of fatty acid synthesis from pyruvate due to the high ratio of acetyl-CoA against CoA was maintained by the action of the oxygen-sensitive pyruvate dehydrogenase found in Euglena mitochondria. When [2-14C]pyruvate was fed into intact mitochondria under anaerobic conditions, radioactive fatty acids were formed in the presence of malate, which provided reducing power for the matrix.     

Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short chain fatty acids

Short chain fatty acids such as acetic, propionic, and butyric acids can be synthesized into polyhydroxyalkanoates (PHAs) by Ralstonia eutropha. Metabolic carbon fluxes of the acids in living cells have significant effect on the yield, composition, and thermomechanical properties of PHA bioplastics. Based on the general knowledge of central metabolism pathways and the unusual metabolic pathways in R. eutropha, a metabolic network of 41 bioreactions is constructed to analyze the carbon fluxes on utilization of the short chain fatty acids. In fed-batch cultures with constant feeding of acid media, carbon metabolism and distribution in R. eutropha were measured involving CO2, PHA biopolymers, and residual cell mass. As the cells underwent unsteady state metabolism and PHA biosynthesis under nitrogen-limited conditions, accumulative carbon balance was applied for pseudo-steady-state analysis of the metabolic carbon fluxes. Cofactor NADP/NADPH balanced between PHA synthesis and the C3/C4 pathway provided an independent constraint for solution of the underdetermined metabolic network. A major portion of propionyl-CoA was directed to pyruvate via the 2-methylcitrate cycle and further decarboxylated to acetyl-CoA. Only a small amount of propionate carbon (<15% carbon) was directly condensed with acetyl-CoA for 3-hydroxyvalerate. The ratio of glyoxylate shunt to TCA cycle varies from 0 to 0.25, depending on the intracellular acetyl-CoA level and acetic acid in the medium. Malate is the node of the C3/C4 pathway and TCA cycle and its decarboxylation to dehydrogenation ranges from 0.33 to 1.28 in response to the demands on NADPH and oxaloacetate for short chain fatty acids utilization.     

In vitro lipid metabolism in the rat pancreas. I. Basal lipid metabolism.

1. The in vitro basal lipid metabolism of rat pancreatic fragments was compared with that in adipose tissue fragments and liver slices. 2. [1-14C]Acetate added to the media was mostly incorporated into palmitic acid and to a lesser extent into oleic acid. In addition, pancreatic tissue exhibited a marked capacity for elongation of polyunsaturated fatty acids by [1-14C]acetate and resulting desaturation when compared to adipose tissue and liver. 3. Data obtained in the presence of [U-14C]glucose, [1-14C]palmitate and 3H20 indicate that acetyl-CoA derived from glucose and from beta-oxidation of fatty acids contributed to de novo lipogenesis. 4. Oxidation of [1-14C]palmitic acid was 9-13 times higher in the pancreas than in adipose tissue or liver when expressed on a wet weight basis. 5. The fatty acid moiety of pancreatic glycerolipids could be derived from de novo synthesis, fatty acids added to the medium, or from fatty acids formed from the hydrolysis of endogenous lipids. The glycerol moiety could be derived either from glucose, or directly from glycerol through participation of glycerol kinase.     

Peroxisomal fatty acid oxidation is a substantial source of the acetyl moiety of malonyl-CoA in rat heart

Little is known about the sources of acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of mitochondrial fatty acid oxidation in the heart. In perfused rat hearts, we previously showed that malonyl-CoA is labeled from both carbohydrates and fatty acids. This study was aimed at assessing the mechanisms of incorporation of fatty acid carbons into malonyl-CoA. Rat hearts were perfused with glucose, lactate, pyruvate, and a fatty acid (palmitate, oleate or docosanoate). In each experiment, substrates were (13)C-labeled to yield singly or/and doubly labeled acetyl-CoA. The mass isotopomer distribution of malonyl-CoA was compared with that of the acetyl moiety of citrate, which reflects mitochondrial acetyl-CoA. In the presence of labeled glucose or lactate/pyruvate, the (13)C labeling of malonyl-CoA was up to 2-fold lower than that of mitochondrial acetyl-CoA. However, in the presence of a fatty acid labeled in its first acetyl moiety, the (13)C labeling of malonyl-CoA was up to 10-fold higher than that of mitochondrial acetyl-CoA. The labeling of malonyl-CoA and of the acetyl moiety of citrate is compatible with peroxisomal beta-oxidation forming C(12) and C(14) acyl-CoAs and contributing >50% of the fatty acid-derived acetyl groups that end up in malonyl-CoA. This fraction increases with the fatty acid chain length. By supplying acetyl-CoA for malonyl-CoA synthesis, peroxisomal beta-oxidation may participate in the control of mitochondrial fatty acid oxidation in the heart. In addition, this pathway may supply some acyl groups used in protein acylation, which is increasingly recognized as an important regulatory mechanism for many biochemical processes.     

Effects of clofibrate feeding on essential fatty acid desaturation and oxidation in isolated rat liver cells.

The effects of clofibrate feeding on the metabolism of polyunsaturated fatty acids were studied in isolated rat hepatocytes. Administration of clofibrate stimulated the oxidation and particularly the peroxisomal beta-oxidation of all the fatty acids used. The increase in oxidation products was markedly higher when n-3 fatty acids were used as substrate, indicating that peroxisomes contribute more to the oxidation of n-3 than n-6 fatty acids. The whole increase in oxidation could be accounted for by a corresponding decrease in acylation in triacylglycerol while the esterification in phospholipids remained unchanged. A marked stimulation of the amounts of newly synthesized C16 and C18 fatty acids recovered, was observed when 18:2(n-6), 20:3(n-6), 18:3 (n-3) and 20:5(n-3), but not when 20:4(n-6) and 22:4(n-6) were used as substrate. This agrees with the view that extra-mitochondrial acetyl-CoA produced from peroxisomal beta-oxidation is more easily used for fatty acid new synthesis than acetyl-CoA from mitochondrial beta-oxidation. The delta 6 and delta 5 desaturase activities were distinctly higher in cells from clofibrate fed rats indicating a stimulating effect.     

FATP1 mediates fatty acid-induced activation of AMPK in 3T3-L1 adipocytes

Fatty acid transport proteins are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. FATP-dependent production of AMP was evaluated using FATP4 proteoliposomes, and fatty acid-dependent activation of AMP-activated protein kinase (AMPK) was assessed in 3T3-L1 adipocytes. Insulin-stimulated fatty acid influx (palmitate or arachidonate) into cultured adipocytes resulted in an increase in the phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. Consistent with the activation of AMPK, palmitate uptake into 3T3-L1 adipocytes resulted in an increase in intracellular [AMP]/[ATP]. The fatty acid-induced increase in AMPK activation was attenuated in a cell line expressing shRNA targeting FATP1. Taken together, these results demonstrate that, in adipocytes, insulin-stimulated fatty acid influx mediated by FATP1 regulates AMPK and provides a potential regulatory mechanism for balancing de novo production of fatty acids from glucose metabolism with influx of preformed fatty acids via phosphorylation of acetyl-CoA carboxylase.     

Effect of acetate formation pathway and long chain fatty acid CoA-ligase on the free fatty acid production in E. coli expressing acy-ACP thioesterase from Ricinus communis

Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells.     

Metabolic adaptations during lactogenesis. Fatty acid synthesis in rabbit mammary tissue during pregnancy and lactation

1. Mammary tissue was obtained from rabbits at various stages of pregnancy and lactation and used for tissue-slice incubations (to measure the rate of fatty acid synthesis and CO(2) production) and to determine relevant enzymic activities. A biphasic adaptation in fatty acid synthetic capacity during lactogenesis was noted. 2. The first lactogenic response occurred between day 15 and 24 of pregnancy. Over this period fatty acid synthesis (from acetate) increased 14-fold and the proportions of fatty acids synthesized changed to those characteristic of milk fat (77-86% as C(8:0)+C(10:0) acids). 3. The second lactogenic response occurred post partum as indicated by increased rates of fatty acid synthesis and CO(2) production (from acetate and glucose) and increased enzymic activities. 4. Major increases in enzymic activities between mid-pregnancy and lactation were noted for ATP citrate lyase (EC 4.1.3.8), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA carboxylase (EC 6.4.1.2), fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Smaller increases in activity occurred with glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) and NADP(+)-isocitrate dehydrogenase (EC 1.1.1.42) and the activity of NADP(+)-malate dehydrogenase (EC 1.1.1.40) was negligible at all periods tested. 5. During pregnancy and lactation there was a close temporal relationship between fatty acid synthetic capacity and the activities of ATP citrate lyase (r=0.94) and acetyl-CoA carboxylase (r=0.90).     

Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo

1. The extractions of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo were calculated from measurements of their arterial and coronary sinus blood concentration. Elevation of plasma free fatty acid concentrations by infusion of intralipid and heparin resulted in increased extraction of free fatty acids and diminished extractions of glucose, lactate and pyruvate by the heart. It is suggested that metabolism of free fatty acids by the heart in vivo, as in vitro, may impair utilization of these substrates. These effects of elevated plasma free fatty acid concentrations on extractions by the heart in vivo were reversed by injection of dichloroacetate, which also improved extraction of lactate and pyruvate by the heart in vivo in alloxan diabetes. 2. Sodium dichloroacetate increased glucose oxidation and pyruvate oxidation in hearts from fed normal or alloxan-diabetic rats perfused with glucose and insulin. Dichloroacetate inhibited oxidation of acetate and 3-hydroxybutyrate and partially reversed inhibitory effects of these substrates on the oxidation of glucose. In rat diaphragm muscle dichloroacetate inhibited oxidation of acetate, 3-hydroxybutyrate and palmitate and increased glucose oxidation and pyruvate oxidation in diaphragms from alloxan-diabetic rats. Dichloroacetate increased the rate of glycolysis in hearts perfused with glucose, insulin and acetate and evidence is given that this results from a lowering of the citrate concentration within the cell, with a consequent activation of phosphofructokinase. 3. In hearts from normal rats perfused with glucose and insulin, dichloroacetate increased cell concentrations of acetyl-CoA, acetylcarnitine and glutamate and lowered those of aspartate and malate. In perfusions with glucose, insulin and acetate, dichloroacetate lowered the cell citrate concentration without lowering the acetyl-CoA or acetylcarnitine concentrations. Measurements of specific radioactivities of acetyl-CoA, acetylcarnitine and citrate in perfusions with [1-(14)C]acetate indicated that dichloroacetate lowered the specific radio-activity of these substrates in the perfused heart. Evidence is given that dichloroacetate may not be metabolized by the heart to dichloroacetyl-CoA or dichloroacetylcarnitine or citrate or CO(2). 4. We suggest that dichloroacetate may activate pyruvate dehydrogenase, thus increasing the oxidation of pyruvate to acetyl-CoA and acetylcarnitine and the conversion of acetyl-CoA into glutamate, with consumption of aspartate and malate. Possible mechanisms for the changes in cell citrate concentration and for inhibitory effects of dichloroacetate on the oxidation of acetate, 3-hydroxybutyrate and palmitate are discussed.     

Coupling of Metabolism and Bioconversion: Microbial Esterification of Citronellol with Acetyl Coenzyme A Produced via Metabolism of Glucose in an Interface Bioreactor

Microbial esterification of citronellol with acetyl coenzyme A (acetyl-CoA) produced via metabolism of glucose was performed with Hansenula saturnus IFO 0809 in an interface bioreactor by using a hydrophilic carrier (an agar plate or an agar-coated filter pad) and a hydrophobic organic solvent (decane). An increase in the glucose concentration on an agar plate led to a decrease in oxidation of citronellol and an increase in the amount of citronellyl acetate. Fed-batch addition of citronellol efficiently alleviated substrate toxicity and resulted in the accumulation of high levels of citronellyl acetate. Furthermore, triple coupling of acetyl-CoA formation, microbial reduction of citronellal to citronellol, and esterification of the resulting citronellol with acetyl-CoA was also an efficient way to prepare citronellyl acetate. By using coupling systems, citronellyl acetate could be efficiently produced without any acetyl donor.     

葡萄糖和维生素C对普安银鲫卵黄囊仔鱼ACC、FAS及CPTⅠ活性的影响

本试验旨在探究普安银鲫(Carassius auratus )卵黄囊仔鱼发育过程中ACC、FAS及CPT I活性变化及葡萄糖和维生素C溶液分别浸泡对它们的影响。采用酶学方法研究了普安银鲫卵黄囊仔鱼过程中ACC、FAS及CPT I活性变化的变化特点。结果显示：在卵黄囊仔鱼发育过程中,对照组与维生素C组中ACC和FAS活性呈上升趋势,CPT I活性呈“下降-上升”变化趋势,而葡萄糖组ACC、FAS及CPT I活性均呈上升趋势,且3种酶的活性均显著高于对照组(P<0.05)。维生素C组ACC活性在内源营养期显著高于对照组,FAS活性在混合营养期和外源营养期显著高于对照组,CPT I活性在内源营养期和外源营养期显著高于对照组(P<0.05)。研究表明：ACC、FAS及CPT I在维持普安银鲫卵黄囊仔鱼发育中脂质代谢的动态平衡起着重要作用,15g/L的葡萄糖溶液可通过调节仔鱼体内脂质代谢酶的活性而形成新的脂质代谢水平,以满足仔鱼生长发育需要;而30mg/L的维生素C对维持仔鱼发育中体内正常的脂质代谢具有重要作用。