Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos

The reactions leading to triacylglycerol (TAG) synthesis in oilseeds have been well characterized. However, quantitative analyses of acyl group and glycerol backbone fluxes that comprise extraplastidic phospholipid and TAG synthesis, including acyl editing and phosphatidylcholine-diacylglycerol interconversion, are lacking. To investigate these fluxes, we rapidly labeled developing soybean (Glycine max) embryos with [¹⁴C]acetate and [¹⁴C]glycerol. Cultured intact embryos that mimic in planta growth were used. The initial kinetics of newly synthesized acyl chain and glycerol backbone incorporation into phosphatidylcholine (PC), 1,2-sn-diacylglycerol (DAG), and TAG were analyzed along with their initial labeled molecular species and positional distributions. Almost 60% of the newly synthesized fatty acids first enter glycerolipids through PC acyl editing, largely at the sn-2 position. This flux, mostly of oleate, was over three times the flux of nascent [¹⁴C]fatty acids incorporated into the sn-1 and sn-2 positions of DAG through glycerol-3-phosphate acylation. Furthermore, the total flux for PC acyl editing, which includes both nascent and preexisting fatty acids, was estimated to be 1.5 to 5 times the flux of fatty acid synthesis. Thus, recycled acyl groups (16:0, 18:1, 18:2, and 18:3) in the acyl-coenzyme A pool provide most of the acyl chains for de novo glycerol-3-phosphate acylation. Our results also show kinetically distinct DAG pools. DAG used for TAG synthesis is mostly derived from PC, whereas de novo synthesized DAG is mostly used for PC synthesis. In addition, two kinetically distinct sn-3 acylations of DAG were observed, providing TAG molecular species enriched in saturated or polyunsaturated fatty acids.     

High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [¹³C₁₈]oleic acid. The [¹³C₁₈]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [¹³C₁₈]oleic acid to rats. The [¹³C₁₈]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [¹³C₁₈]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [¹³C₁₈]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [¹³C₁₈]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.     

Evidence for Transaldolase Activity in the Isolated Heart Supplied with [U-13C3]Glycerol

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-¹³C₃]glycerol, and ²H₂O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, ¹³C from [U-¹³C₃]glycerol appeared in glycogen in carbon positions 4–6 of glucosyl units but none in positions 1–3. The extent of [4,5,6-¹³C₃]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H₂O₂. Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked ¹³C asymmetry in glycogen can only be attributed to conversion of [U-¹³C₃]glycerol to [U-¹³C₃]dihydroxyacetone phosphate and [U-¹³C₃]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this ¹³C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.     

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions

Mixotrophic cultivation can increase microalgae productivity, yet the associated lipid metabolism remains mostly unknown. Stable isotope labeling was used to track assimilation of glycerol into the triacylglyceride (TAG) and membrane lipids of Nannochloropsis salina. In N-replete media, glycerol uptake and ¹³C incorporation into acyl chains were, respectively, 6-fold and 12-fold higher than in N-deplete conditions. In N-replete cultures, 42% of the carbon in the consumed glycerol was assimilated into lipid acyl chains, mostly in membrane lipids rather than TAG. In N-deplete cultures, only 11% of the limited amount of consumed glycerol was fixed into lipid acyl chains. Labeled lipid-associated glycerol backbones were predominantly ¹³C₃ labeled, suggesting that intact glycerol molecules were directly esterified with fatty acids/polar head groups. However, the presence of singly and doubly labeled lipid-bound glycerol species suggested that some glycerol also went through the central carbon metabolism before forming glycerol-3-phosphate destined for lipid esterification. ¹³C incorporation was higher in the saturated and monounsaturated than the polyunsaturated acyl chains of TAG, indicating the flux of carbon from glycerol went first to de novo fatty acid synthesis before acyl editing reactions. The results demonstrate that nitrogen availability influences both glycerol consumption and utilization for lipid synthesis in Nannochloropsis, providing novel insights for developing mixotrophic cultivation strategies.     

Berberine treatment attenuates the palmitate-mediated inhibition of glucose uptake and consumption through increased 1,2,3-triacyl-sn-glycerol synthesis and accumulation in H9c2 cardiomyocytes

Dysfunction of lipid metabolism and accumulation of 1,2-diacyl-sn-glycerol (DAG) may be a key factor in the development of insulin resistance in type 2 diabetes. Berberine (BBR) is an isoquinoline alkaloid extract that has shown promise as a hypoglycemic agent in the management of diabetes in animal and human studies. However, its mechanism of action is not well understood. To determine the effect of BBR on lipid synthesis and its relationship to insulin resistance in H9c2 cardiomyocytes, we measured neutral lipid and phospholipid synthesis and their relationship to glucose uptake. Compared with controls, BBR treatment stimulated 2-[1,2-³H(N)]deoxy-D-glucose uptake and consumption in palmitate-mediated insulin resistant H9c2 cells. The mechanism was though an increase in protein kinase B (AKT) activity and GLUT-4 glucose transporter expression. DAG accumulated in palmitate-mediated insulin resistant H9c2 cells and treatment with BBR reduced this DAG accumulation and increased accumulation of 1,2,3-triacyl-sn-glycerol (TAG) compared to controls. Treatment of palmitate-mediated insulin resistant H9c2 cells with BBR increased [1,3-³H]glycerol and [1-¹⁴C]glucose incorporation into TAG and reduced their incorporation into DAG compared to control. In addition, BBR treatment of these cells increased [1-¹⁴C]palmitic acid incorporation into TAG and decreased its incorporation into DAG compared to controls. BBR treatment did not alter phosphatidylcholine or phosphatidylethanolamine synthesis. The mechanism for the BBR-mediated decreased precursor incorporation into DAG and increased incorporation into TAG in palmitate-incubated cells was an increase in DAG acyltransferase-2 activity and its expression and a decrease in TAG hydrolysis. Thus, BBR treatment attenuates palmitate-induced reduction in glucose uptake and consumption, in part, through reduction in cellular DAG levels and accumulation of TAG in H9c2 cells.     

Phospholipid metabolism in boar spermatozoa and role of diacylglycerol species in the De Novo formation of phosphatidylcholine

We have investigated pathways of lipid metabolism in boar spermatozoa sperm cells incubated for up to 3 days with [¹⁴C]palmitic acid, [¹⁴C]glycerol, [¹⁴C]choline, or [¹⁴C]arachidonic acid or incorporated these precursors into diglycerides and/or phospholipids. When spermatozoa were incubated with [¹⁴C]palmitic acid or [¹⁴C]glycerol, there was first an incorporation into phosphatidic acid, followed by labelling of 1,2-diacylglycerol (DAG) and then phosphatidyl-choline (PC). This indicates that the de novo pathway of phospholipid synthesis is active in these cells. However, not all DAG was converted to PC. A pool of di-saturated DAG, which represented a considerable proportion of the high basal levels of DAG, accumulated the majority of label. Another DAG pool, containing saturated fatty acids in position 1 and unsaturated fatty acids in position 2 and representing the remaining basal DAG, was in equilibrium with PC. When spermatozoa were incubated with [¹⁴C]arachidonic acid, there was a considerable incorporation of label into PC, which indicates the presence of an active deacylation/reacylation cycle. The behaviour of certain lipid pools varied depending on the temperature at which spermatozoa were incubated. For example, in the presence of [¹⁴C]palmitic acid or [¹⁴C]arachidonic acid, there was more incorporation of label into PC when spermatozoa were incubated at 25°C than when incubated at 17°C. Taken together, these results indicate that spermatozoa have an active lipid synthetic capacity. It may therefore be possible to design methods to evaluate the metabolic activity of boar spermatozoa based on the incorporation of lipid precursors under standardized conditions. Mol. Reprod. Dev. 47:105–112, 1997. © 1997 Wiley-Liss, Inc.     

Metabolism of triacylglycerol in developing rat brain

Metabolism of triacylglycerol (TAG) in developing brain has been examined. TAG is a relatively minor fraction of brain lipid in both suckling and adult rats and cannot be accounted for as entrapped blood. When glycerol tri[1-¹⁴C]oleate and [2-³H]glycerol trioleate were simultaneously injected intracerebrally into suckling rats, both labels appeared in diacylglycerol and the major phospholipids; acyl chain label was incorporated more extensively at early time points, with choline phosphoglycerides being most actively labeled. With [1-¹⁴C]fatty acids and [2-³H] glycerol administration, the specific activity of TAG was much greater than that of the more abundant phospholipids. Although direct acyl exchange between TAG and phospholipids was not demonstrated, relationships of TAG to selective mechanisms of phosphoglyceride synthesis were indicated.Abbreviations used TAG triacylglycerol - DAG diacylcerol - HPLC high performance liquid chromatography - CoA coenzyme A - BSA bovine serum albumin - TLC thin layer chromatography - DPM disintegrations per minute - ATP adenosine triphosphate - GLC gas liquid chromatography - PC choline, phosphoglyceride - PE ethanolamine phosphoglyceride - PS serine phosphoglyceride - PI inositol phosphoglyceride     

Phosphatidylcholine:diacylglycerol cholinephosphotransferase’s unique regulation of castor bean oil quality

Castor bean (Ricinus communis) seed oil (triacylglycerol [TAG]) is composed of ∼90% of the industrially important ricinoleoyl (12-hydroxy-9-octadecenoyl) groups. Here, phosphatidylcholine (PC):diacylglycerol (DAG) cholinephosphotransferase (PDCT) from castor bean was biochemically characterized and compared with camelina (Camelina sativa) PDCT. DAGs with ricinoleoyl groups were poorly used by Camelina PDCT, and their presence inhibited the utilization of DAG with “common” acyl groups. In contrast, castor PDCT utilized DAG with ricinoleoyl groups similarly to DAG with common acyl groups and showed a 10-fold selectivity for DAG with one ricinoleoyl group over DAG with two ricinoleoyl groups. Castor DAG acyltransferase2 specificities and selectivities toward different DAG and acyl-CoA species were assessed and shown to not acylate DAG without ricinoleoyl groups in the presence of ricinoleoyl-containing DAG. Eighty-five percent of the DAG species in microsomal membranes prepared from developing castor endosperm lacked ricinoleoyl groups. Most of these species were predicted to be derived from PC, which had been formed by PDCT in exchange with DAG with one ricinoleoyl group. A scheme of the function of PDCT in castor endosperm is proposed where one ricinoleoyl group from de novo-synthesized DAG is selectivity transferred to PC. Nonricinoleate DAG is formed and ricinoleoyl groups entering PC are re-used either in de novo synthesis of DAG with two ricinoleoyl groups or in direct synthesis of triricinoleoyl TAG by PDAT. The PC-derived DAG is not used in TAG synthesis but is proposed to serve as a substrate in membrane lipid biosynthesis during oil deposition.

The enzyme phosphatidylcholine:diacylglycerol cholinephosphotransferase facilitates accumulation of seed oil with three hydroxylated acyl groups in Ricinus communis.     

Interaction between the Pentose Phosphate Pathway and Gluconeogenesis from Glycerol in the Liver

After exposure to [U-¹³C₃]glycerol, the liver produces primarily [1,2,3-¹³C₃]- and [4,5,6-¹³C₃]glucose in equal proportions through gluconeogenesis from the level of trioses. Other ¹³C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex ¹³C-labeling patterns in exported glucose. Here, we investigated ¹³C labeling in plasma glucose in rats given [U-¹³C₃]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a ¹³C-¹³C bond within the trioses contributing to glucose, the appearance of [1,2-¹³C₂]-, [2,3-¹³C₂]-, [5,6-¹³C₂]-, and [4,5-¹³C₂]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-¹³C₂]glucose/[2,3-¹³C₂]glucose was significantly greater than [5,6-¹³C₂]glucose/[4,5-¹³C₂]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by ¹³C distribution in blood glucose after [U-¹³C₃]glycerol administration.     

The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds

Engineering of oilseed plants to accumulate unusual fatty acids (FAs) in seed triacylglycerol (TAG) requires not only the biosynthetic enzymes for unusual FAs but also efficient utilization of the unusual FAs by the host-plant TAG biosynthetic pathways. Competing pathways of diacylglycerol (DAG) and subsequent TAG synthesis ultimately affect TAG FA composition. The membrane lipid phosphatidylcholine (PC) is the substrate for many FA-modifying enzymes (desaturases, hydroxylases, etc.) and DAG can be derived from PC for TAG synthesis. The relative proportion of PC-derived DAG versus de novo synthesized DAG utilized for TAG synthesis, and the ability of each pathway to utilize unusual FA substrates, are unknown for most oilseed plants, including Arabidopsis thaliana. Through metabolic labeling experiments we demonstrate that the relative flux of de novo DAG into the PC-derived DAG pathway versus direct conversion to TAG is ～14/1 in wild-type Arabidopsis. Expression of the Ricinus communis FA hydroxylase reduced the flux of de novo DAG into PC by ～70%. Synthesis of TAG directly from de novo DAG did not increase, resulting in lower total synthesis of labeled lipids. Hydroxy-FA containing de novo DAG was rapidly synthesized, but it was not efficiently accumulated or converted to PC and TAG, and appeared to be in a futile cycle of synthesis and degradation. However, FA hydroxylation on PC and conversion to DAG allowed some hydroxy-FA to accumulate in sn-2 TAG. Therefore, the flux of DAG through PC represents a major bottleneck for the accumulation of unusual FAs in TAG of transgenic Arabidopsis seeds.     

Alterations of fatty acyl turnover in macrophage glycerolipids induced by stimulation. Evidence for enhanced recycling of arachidonic acid

Glycerophospholipid biosynthesis by the de novo pathway was assessed in mouse peritoneal macrophages by pulse-labeling with [U-¹⁴C]glycerol. Phosphatidylcholine (PC), which amounts to about 35% of total cellular phospholipids, exhibited the highest rate of glycerol uptake, followed by phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Remodeling of PC molecular species by deacylation/reacylation was established by determining the redistribution of glycerol label over 2 h after a 1 h pulse of [U-¹⁴C]glycerol and by determining incorporation of ¹⁸O from H₂ ¹⁸O-containing media. These data suggest that stearic and arachidonic acid enter PC primarily by the remodeling pathway but that small amounts of highly unsaturated molecular species, including 1,2-diarachidonoyl PC, are rapidly synthesized de novo, and subsequently remodeled or degraded. Treatment of the cells with the ionophore A23187 resulted in the selective enhancement of arachidonate turnover in PC, PI and neutral lipid, as well as enhanced de novo PI synthesis. [U-¹⁴C]Glycerol labeling experiments suggest that arachidonic acid liberated by Ca²⁺-dependent phospholipase A₂ activity is also reacylated in part through de novo glycerolipid biosynthesis, leading to the formation and remodeling of 1,2-diarachidonoyl PC and other highly polyunsaturated molecular species.     

Isotopologue Profiling of Legionella pneumophila: ROLE OF SERINE AND GLUCOSE AS CARBON SUBSTRATES*

Legionella pneumophila (Lp) is commonly found in freshwater habitats but is also the causative agent of Legionnaires'' disease when infecting humans. Although various virulence factors have been reported, little is known about the nutrition and the metabolism of the bacterium. Here, we report the application of isotopologue profiling for analyzing the metabolism of L. pneumophila. Cultures of Lp were supplied with [U-¹³C₃]serine, [U-¹³C₆]glucose, or [1,2-¹³C₂]glucose. After growth, ¹³C enrichments and isotopologue patterns of protein-derived amino acids and poly-3-hydroxybutyrate were determined by mass spectrometry and/or NMR spectroscopy. The labeling patterns detected in the experiment with [U-¹³C₃]serine showed major carbon flux from serine to pyruvate and from pyruvate to acetyl-CoA, which serves as a precursor of poly-3-hydroxybutyrate or as a substrate of a complete citrate cycle with Si specificity of the citrate synthase. Minor carbon flux was observed between pyruvate and oxaloacetate/malate by carboxylation and decarboxylation, respectively. The apparent lack of label in Val, Ile, Leu, Pro, Phe, Met, Arg, and Tyr confirmed that L. pneumophila is auxotrophic for these amino acids. Experiments with [¹³C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-¹³C₂]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (Δzwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp.     

Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of<Emphasis Type="Italic"> Brassica napus</Emphasis> L.<Emphasis Type="Italic"> in planta</Emphasis>

Glycerol-3-phosphate (glycerol-3P) is a primary substrate for triacylglycerol synthesis. In the present study, changes in the levels of glycerol-3P during rape (Brassica napus L.) seed development and the influence of manipulating glycerol-3P levels on triacylglycerol synthesis were investigated. (i) Glycerol-3P levels were high in young seeds and decreased during seed development at 30 and 40 days after flowering (DAF), when lipid accumulation was maximal. (ii) To manipulate glycerol-3P levels in planta, various concentrations of glycerol were injected directly into 30-DAF seeds, which remained otherwise intact within their siliques and attached to the plant. Injection of 0–10 nmol glycerol led to a progressive increase in seed glycerol-3P levels within 28 h. (iii). Increased levels of glycerol-3P were accompanied by an increase in the flux of injected [¹⁴C]sucrose into total lipids and triacylglycerol, whereas fluxes to organic acids, amino acids, starch, protein and cell walls were not affected. (iv) When [¹⁴C]acetate was injected into seeds, label incorporation into total lipids and triacylglycerol increased progressively with increasing glycerol-3P levels. (v) There was a strong correlation between the level of glycerol-3P and the incorporation of injected [¹⁴C]acetate and [¹⁴C]sucrose into triacylglycerol. (v) The results provide evidence that the prevailing levels of glycerol-3P co-limit triacylglycerol synthesis in developing rape seeds.Abbreviations DAF Days after flowering - DAG Diacylglycerol - G3PAT Glycerol-3-phosphate acyltransferase - Glycerol-3P Glycerol-3-phosphate - PA Phosphatidic acid - PC Phosphatidylcholine - TAG Triacylglycerol,     

An LC/MS/MS method for stable isotope dilution studies of β-carotene bioavailability,bioconversion, and vitamin A status in humans

Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as β-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [¹³C₁₀]β-carotene and 1 mg [¹³C₁₀]retinyl acetate in human subjects over a 2 week period. A reverse phase C₁₈ column and binary mobile phase solvent system separated β-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537→321 and m/z 269→93 for respective [¹²C]β-carotene and [¹²C] retinoids; m/z 547→330 and m/z 274→98 for [¹³C₁₀]β-carotene and [¹³C₅] cleavage products; and m/z 279→100 for metabolites of [¹³C₁₀]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [¹³C₁₀]retinyl acetate with [¹³C₁₀]β-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual''s vitamin A status.     

The role of light in soybean seed filling metabolism

Soybean (Glycine max) yields high levels of both protein and oil, making it one of the most versatile and important crops in the world. Light has been implicated in the physiology of developing green seeds including soybeans but its roles are not quantitatively understood. We have determined the light levels reaching growing soybean embryos under field conditions and report detailed redox and energy balance analyses for them. Direct flux measurements and labeling patterns for multiple labeling experiments including [U‐¹³C₆]‐glucose, [U‐¹³C₅]‐glutamine, the combination of [U‐¹⁴C₁₂]‐sucrose + [U‐¹⁴C₆]‐glucose + [U‐¹⁴C₅]‐glutamine + [U‐¹⁴C₄]‐asparagine, or ¹⁴CO₂ labeling were performed at different light levels to give further insight into green embryo metabolism during seed filling and to develop and validate a flux map. Labeling patterns (protein amino acids, triacylglycerol fatty acids, starch, cell wall, protein glycan monomers, organic acids), uptake fluxes (glutamine, asparagine, sucrose, glucose), fluxes to biomass (protein amino acids, oil), and respiratory fluxes (CO₂, O₂) were established by a combination of gas chromatography‐mass spectrometry, ¹³C‐ and ¹H‐NMR, scintillation counting, HPLC, gas chromatography‐flame ionization detection, C:N and amino acid analyses, and infrared gas analysis, yielding over 750 measurements of metabolism. Our results show: (i) that developing soybeans receive low but significant light levels that influence growth and metabolism; (ii) a role for light in generating ATP but not net reductant during seed filling; (iii) that flux through Rubisco contributes to carbon conversion efficiency through generation of 3‐phosphoglycerate; and (iv) a larger contribution of amino acid carbon to fatty acid synthesis than in other oilseeds analyzed to date.     

Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, ¹³C metabolic flux analysis with parallel studies on [1-¹³C^Fru]sucrose, [1-¹³C^Glc]sucrose, and [¹³C₆^Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS^Man or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Neutral lipid storage disease: a possible functional defect in phospholipid-linked triacylglycerol metabolism

Neutral lipid storage disease (NLSD) (Chanarin-Dorfman Syndrome) is an autosomal recessive disorder of multisystem triacyglycerol (TAG) storage. Previous work has pointed to a defect in intracellular TAG metabolism. In the studies reported here, the lipid metabolism of three lines of NLSD fibroblasts were compared to normal skin fibroblasts. When pulsed with [³H]oleic acid, the earliest observed abnormality in NLSD cell lines was increased incorporation into phosphatidylethanolamine, followed by accumulation of radiolabel in TAG. Activities of several glycerolipid synthetic enzymes were comparable in NLSD and normal fibroblast lines, excluding oversynthesis of glycerolipid. The proportion of plasmalogen and neutral ether lipid synthesized was normal and alkylglycerols did not accumulate, excluding a defect in ether lipid metabolism. Activities of both acid lipase and Mn²⁺-sensitive lipase within the particulate fractions of NLSD and normal fibroblasts were comparable. These studies are most consistent with functional deficiency of a TAG lipase with activity against a pool of TAG that are normally utilized for phospholipid biosynthesis.     

para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable ¹³C₆-isotope of pABA (p- amino[aromatic-¹³C₆]benzoic acid ([¹³C₆]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[¹³C₆]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [¹³C₆]pABA or [¹³C₆]4HB generate both ¹³C₆-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product ¹³C₆-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ₆. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ₆ quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.