Triose Phosphates Research Articles

D‐Lactate production as a function of glucose metabolism in Saccharomyces cerevisiae

Methylglyoxal, a reactive, toxic dicarbonyl, is generated by the spontaneous degradation of glycolytic intermediates. Methylglyoxal can form covalent adducts with cellular macromolecules, potentially disrupting cellular function. We performed experiments using the model organism Saccharomyces cerevisiae, grown in media containing low, moderate and high glucose concentrations, to determine the relationship between glucose consumption and methylglyoxal metabolism. Normal growth experiments and glutathione depletion experiments showed that metabolism of methylglyoxal by log-phase yeast cultured aerobically occurred primarily through the glyoxalase pathway. Growth in high-glucose media resulted in increased generation of the methylglyoxal metabolite D-lactate and overall lower efficiency of glucose utilization as measured by growth rates. Cells grown in high-glucose media maintained higher glucose uptake flux than cells grown in moderate-glucose or low-glucose media. Computational modelling showed that increased glucose consumption may impair catabolism of triose phosphates as a result of an altered NAD⁺:NADH ratio.

Role of metabolite transporters in source-sink carbon allocation

Plants assimilate carbon dioxide during photosynthesis in chloroplasts. Assimilated carbon is subsequently allocated throughout the plant. Generally, two types of organs can be distinguished, mature green source leaves as net photoassimilate exporters, and net importers, the sinks, e.g., roots, flowers, small leaves, and storage organs like tubers. Within these organs, different tissue types developed according to their respective function, and cells of either tissue type are highly compartmentalized. Photoassimilates are allocated to distinct compartments of these tissues in all organs, requiring a set of metabolite transporters mediating this intercompartmental transfer. The general route of photoassimilates can be briefly described as follows. Upon fixation of carbon dioxide in chloroplasts of mesophyll cells, triose phosphates either enter the cytosol for mainly sucrose formation or remain in the stroma to form transiently stored starch which is degraded during the night and enters the cytosol as maltose or glucose to be further metabolized to sucrose. In both cases, sucrose enters the phloem for long distance transport or is transiently stored in the vacuole, or can be degraded to hexoses which also can be stored in the vacuole. In the majority of plant species, sucrose is actively loaded into the phloem via the apoplast. Following long distance transport, it is released into sink organs, where it enters cells as source of carbon and energy. In storage organs, sucrose can be stored, or carbon derived from sucrose can be stored as starch in plastids, or as oil in oil bodies, or – in combination with nitrogen – as protein in protein storage vacuoles and protein bodies. Here, we focus on transport proteins known for either of these steps, and discuss the implications for yield increase in plants upon genetic engineering of respective transporters.

Fructose Degradation in the Haloarchaeon Haloferax volcanii Involves a Bacterial Type Phosphoenolpyruvate-Dependent Phosphotransferase System, Fructose-1-Phosphate Kinase, and Class II Fructose-1,6-Bisphosphate Aldolase

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea.

The role of glyoxalases for sugar stress and aging, with relevance for dyskinesia, anxiety, dementia and Parkinson's disease

Carbohydrates are the primordial source of energy and carbon for certain unicellular organisms as well as for the complex mammalian brain and its synaptic functions [1]. However, for all these cells the degradation of carbohydrates poses several problems, e.g. the formation of toxic by-products such as the glycating electrophile methylglyoxal (MG, also named 2-oxo-propanal) or the excessive generation of lactic acid with ensuing pH change [2]. Under conditions of high carbon flux and under limited availability of NAD+, e.g. during anaerobic glycolysis [3], the triose phosphates dihydroxyacetone phosphate and glyceraldehyde-3-phosphate spontaneously decompose to MG, a compound known to contribute to the generation of advanced glycation endproducts (AGEs) [4] – with possibly irreversible damage to lipids, nucleic acids and proteins, in particular of mitochondria [5] – and to the induction of ubiquitin conjugates [6]. In a pathway with very high conservation during evolution, all these cells use the enzyme glyoxalase I (lactoylglutathione lyase) in the presence of glutathione (GSH) to convert MG into S-lactoyl-glutathione (SLG) and then use glyoxalase II (hydroxyacylglutathione hydrolase) to liberate D-lactate and glutathione. In spite of this toxicity of MG, some bacteria use the enzyme methylglyoxal synthase to generate MG, apparently to regulate carbon flux and growth rate [7]. Furthermore, the ratio between GSH and SLG in such bacteria modulates potassium efflux and intracellular acidification [8]. Acting as a signal initiator, MG activates the osmosensor Sln1, the HOG-MAP kinase cascade and the calcium(2+) signalling pathway in yeast [9]. In human cells, MG has a well established anti-proliferative effect in cancerous cells with enhanced glycolysis (Warburg-effect) [10, 11]. MG was recently reported to play a physiological role in the modulation of hypoxia-induced-factor-1α (HIF-1alpha) levels and thus in the modulation of the balance between anaerobic and aerobic bioenergetics [12]. Interestingly, an anxiety-suppressing effect of MG infusions into the brain was observed in mice [13]. And in patients with ditary dyskinesias, episodic disorders of spontaneous movement, it is now known that either defects in the cerebral glucose transport or defects in the putative neuronal SLG sensor protein MR-1 can be responsible for these symptoms [14,15]. These observations suggest that MG and SLG are not simply toxic by-products to be eliminated, but might play an important physiological function in bioenergetic signaling.

Overexpression of Glyoxalase-I Reduces Hyperglycemia-induced Levels of Advanced Glycation End Products and Oxidative Stress in Diabetic Rats

The reactive advanced glycation end product (AGE) precursor methylglyoxal (MGO) and MGO-derived AGEs are associated with diabetic vascular complications and also with an increase in oxidative stress. Glyoxalase-I (GLO-I) transgenic rats were used to explore whether overexpression of this MGO detoxifying enzyme reduces levels of AGEs and oxidative stress in a rat model of diabetes. Rats were made diabetic with streptozotocin, and after 12 weeks, plasma and multiple tissues were isolated for analysis of AGEs, carbonyl stress, and oxidative stress. GLO-I activity was significantly elevated in multiple tissues of all transgenic rats compared with wild-type (WT) littermates. Streptozotocin treatment resulted in a 5-fold increase in blood glucose concentrations irrespective of GLO-I overexpression. Levels of MGO, glyoxal, 3-deoxyglucosone, AGEs, and oxidative stress markers nitrotyrosine, malondialdehyde, and F2-isoprostane were elevated in the diabetic WT rats. In diabetic GLO-I rats, glyoxal and MGO composite scores were significantly decreased by 81%, and plasma AGEs and oxidative stress markers scores were significantly decreased by ∼50%. Hyperglycemia induced a decrease in protein levels of the mitochondrial oxidative phosphorylation complex in the gastrocnemius muscle, which was accompanied by an increase in the lipid peroxidation product 4-hydroxy-2-nonenal, and this was counteracted by GLO-I overexpression. This study shows for the first time in an in vivo model of diabetes that GLO-I overexpression reduces hyperglycemia-induced levels of carbonyl stress, AGEs, and oxidative stress. The reduction of oxidative stress by GLO-I overexpression directly demonstrates the link between glycation and oxidative stress.

Acetyl Radical Production by the Methylglyoxal−Peroxynitrite System: A Possible Route for l-Lysine Acetylation

Methylglyoxal is an α-oxoaldehyde putatively produced in excess from triose phosphates, aminoacetone, and acetone in some disorders, particularly in diabetes. Here, we investigate the nucleophilic addition of ONOO(-), known as a potent oxidant and nucleophile, to methylglyoxal, yielding an acetyl radical intermediate and ultimately formate and acetate ions. The rate of ONOO(-) decay in the presence of methylglyoxal [k(2,app) = (1.0 ± 0.1) × 10(3) M(-1) s(-1); k(2) ≈ 1.0 × 10(5) M(-1) s(-1)] at pH 7.2 and 25 °C was found to be faster than that reported with monocarbonyl substrates (k(2) < 10(3) M(-1) s(-1)), diacetyl (k(2) = 1.0 × 10(4) M(-1) s(-1)), or CO(2) (k(2) = 3-6 × 10(4) M(-1) s(-1)). The pH profile of the methylglyoxal-peroxynitrite reaction describes an ascendant curve with an inflection around pH 7.2, which roughly coincides with the pK(a) values of both ONOOH and H(2)PO(4)(-) ion. Electron paramagnetic resonance spin trapping experiments with 2-methyl-2-nitrosopropane revealed concentration-dependent formation of an adduct that can be attributed to 2-methyl-2-nitrosopropane-CH(3)CO(•) (a(N) = 0.83 mT). Spin trapping with 3,5-dibromo-4-nitrosobenzene sulfonate gave a signal that could be assigned to a methyl radical adduct [a(N) = 1.41 mT; a(H) = 1.35 mT; a(H(m)) = 0.08 mT]. The 2-methyl-2-nitrosopropane-CH(3)CO(•) adduct could also be observed by replacement of ONOO(-) with H(2)O(2), although at much lower yields. Acetyl radicals could be also trapped by added L-lysine as indicated by the presence of (ε)N-acetyl-L-lysine in the spent reaction mixture. This raises the hypothesis that ONOO(-)/H(2)O(2) in the presence of methylglyoxal is endowed with the potential to acetylate proteins in post-translational processes.

The post-illumination chlorophyll fluorescence transient indicates the RuBP regeneration limitation of photosynthesis in low light in Arabidopsis

The mechanism of post-illumination chlorophyll fluorescence transient (PIFT) was investigated in Arabidopsis. PIFT was detected in the wild type after illumination with low light. In the fba3-2 ( fructose-1,6-bisphosphate aldolase) mutant, in which PIFT is enhanced, strong light also induced PIFT. PIFT was suppressed not only in the triose phosphate/phosphate translocator ( tpt-2) mutant, but also in tpt-2 fba3-2, suggesting that triose phosphates, such as dihydroxyacetone phosphate (DHAP), are involved in the PIFT mechanism. We concluded that PIFT is associated with ribulose-1,5-bisphosphate (RuBP)-regeneration limitation of photosynthesis in low light.

Aging, proteotoxicity, mitochondria, glycation, NAD+ and carnosine: possible inter-relationships and resolution of the oxygen paradox

It is suggested that NAD+ availability strongly affects cellular aging and organism lifespan: low NAD+ availability increases intracellular levels of glycolytic triose phosphates (glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate) which, if not further metabolized, decompose spontaneously into methylglyoxal (MG), a glycating agent and source of protein and mitochondrial dysfunction and reactive oxygen species (ROS). MG-damaged proteins and other aberrant polypeptides can induce ROS generation, promote mitochondrial dysfunction and inhibit proteasomal activity. Upregulation of mitogenesis and mitochondrial activity by increased aerobic exercise, or dietary manipulation, helps to maintain NAD+availability and thereby decreases MG-induced proteotoxicity. These proposals can explain the apparent paradox whereby aging is seemingly caused by increased ROS-mediated macromolecular damage but is ameliorated by increased aerobic activity. It is also suggested that increasing mitochondrial activity decreases ROS generation, while excess numbers of inactive mitochondria are deleterious due to increased ROS generation. The muscle- and brain-associated dipeptide, carnosine, is an intracellular buffer which can delay senescence in cultured human fibroblasts and delay aging in senescence-accelerated mice. Carnosine's ability to react with MG and possibly other deleterious carbonyl compounds, and scavenge various ROS, may account for its protective ability towards ischemia and ageing.

Downregulation of pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase activity in sugarcane culms enhances sucrose accumulation due to elevated hexose-phosphate levels

Analyses of transgenic sugarcane clones with 45–95% reduced cytosolic pyrophosphate: d-fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) activity displayed no visual phenotypical change, but significant changes were evident in in vivo metabolite levels and fluxes during internode development. In three independent transgenic lines, sucrose concentrations increased between three- and sixfold in immature internodes, compared to the levels in the wildtype control. There was an eightfold increase in the hexose-phosphate:triose-phosphate ratio in immature internodes, a significant restriction in the triose phosphate to hexose phosphate cycle and significant increase in sucrose cycling as monitored by 13C nuclear magnetic resonance. This suggests that an increase in the hexose-phosphate concentrations resulting from a restriction in the conversion of hexose phosphates to triose phosphates drive sucrose synthesis in the young internodes. These effects became less pronounced as the tissue matured. Decreased expression of PFP also resulted in an increase of the ATP/ADP and UTP/UDP ratios, and an increase of the total uridine nucleotide and, at a later stage, the total adenine nucleotide pool, revealing strong interactions between PPi metabolism and general energy metabolism. Finally, decreased PFP leads to a reduction of PPi levels in older internodes indicating that in these developmental stages PFP acts in the gluconeogenic direction. The lowered PPi levels might also contribute to the absence of increases in sucrose contents in the more mature tissues of transgenic sugarcane with reduced PFP activity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-009-1069-1) contains supplementary material, which is available to authorized users.

The effect of fasting and fructose and glucose infusion on gluconeogenesis and triose phosphate flux in rats in vivo.

Gluconeogenesis was measured quantitatively by mass isotopomer distribution analysis in rats in vivo through determination of the isotopic enrichment of the pool of hepatic triose phosphates, the immediate precursors of glucose, using primarily [2-13C]glycerol infusion after fasting, and following fructose and glucose infusion. The results showed that the liver controls gluconeogenesis, the metabolic sources and disposal of triose phosphates, and the contribution of glucose from glycogen in response to alterations in substrate availability.

Enzymatic prerequisites for ATP formation from triose phosphates in the A 2 and B-cells of the endocrine pancreas of the guinea-pig.

Available data on the metabolism of glucose in the islets of Langerhans are concerned essentially with the B-cells, whereas information on the metabolic properties of the glucagon producing A2-cells is very limited. In this investigation the enzymatic capacity for degradation of triose phosphates has been determined by measurements of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and pyruvate kinase (PK) activities in isolated samples of A2, B- and acinar cells. For GAPDH significant differences were obtained between the cell specimens. The A2-cells showed the highest value, while the activity of acinar cells was lower than that of the B-cells. The same pattern was obtained for PK. The activities of this enzyme were, however, generally lower than for GAPDH. For PGK no differences could be detected between the analyzed specimens, but this enzyme showed a higher activity than the other two enzymes in both the endocrine and exocrine pancreas.

Activities of 6-phosphofructokinases and inorganic pyrophosphatase in aerobic methylotrophic bacteria

The classical radioisotopic and enzymological studies of J.R. Quayle led to the discovery of the ribulosemonophosphate (RuMP) cycle of formaldehyde assimilation in methanotrophs and methylobacteria [1, 2]. The key reactions of the RuMP cycle catalyzed, by 3-hexulosephosphate synthase (HPS) and phospho-3hexuloisomerase, form fructose-6-phosphate from formaldehyde and ribulose-5-phosphate. It was believed that further phosphorylation of fructose-6phosphate to fructose-1,6-bisphosphate is carried out by ATP-dependent 6-phosphofructokinase (ATP-PFK), although its low activity (2‐8 mU/mg protein) in methanotrophs questioned the significance of the glycolytic pathway of phosphosugar transformation in the RuMP cycle. The discovery of the activity of pyrophosphatedependent 6-phosphofructokinase (PP i -PFK) and a high intracellular concentration of inorganic pyrophosphate in three species of methanotrophs employing different pathways of e 1 compound assimilation stimulated the study of the role of PP i and reappraisal of the concepts of the organization and regulation of the RuMP cycle in aerobic methylotrophic bacteria of various taxonomic positions [3]. Along these lines, in this work, the activities of PP i - and ATP-dependent 6-phosphofructokinases and pyrophosphatase (PP i -ase) were determined in a broad spectrum of aerobic methanotrophs and methylobacteria employing various pathways of C 1 assimilation. The work used strains of obligate methanotrophs and methylobacteria from the collection of the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. Cells were harvested in the early stationary phase, as described earlier [3, 4]. A 12-h Escherichia coli K12 culture grown in liquid medium with glucose was also used. Cells were separated from the medium by centrifugation, washed with 50 mM phosphate buffer (pH 7), and destroyed with an IBPM-press in a frozen state. To obtain cell-free extracts, cell homogenates were centrifuged (12000 g , 20 min) and dialyzed against the same buffer. The activities of PP i -PFK (EC 2.7.1.90), ATP-PFK (EC 2.7.1.11), and inorganic pyrophosphatase (EC 3.6.1.1) were determined as described earlier [4]. The data presented are the means of three measurements. The results of determination of the activities of PP i PFK, ATP-PFK, and PP i -ase in obligate and facultative methylobacteria are shown in the table. Since the detected activity of ATP-PFK could in fact be the activity of PP i -PFK at the expense of PP i admixtures, a preparation of yeast inorganic pyrophosphatase (0.2 U/ml) was introduced into the incubation mixture for ATPPFK determination in further experiments. Under these conditions, the ATP-PFK activity was not revealed. The highest PP i -PFK activity was revealed in type I methanotrophs, which assimilate formaldehyde via the RuMP cycle. In type II methanotrophs with the serine pathway, the activity of PP i -PFK was lower than in type I methanotrophs. The reversibility of this reaction and the necessity of the synthesis of hexose phosphates from triose phosphates in type II methanotrophs suggests the involvement of PP i -PFK in the gluconeogenesis of these bacteria. The lowest values of the PP i -PFK activity were recorded in type X methanotrophs of the genera Methylococcus and Methylocaldum , which simultaneously employ three pathways of C 1 assimilation. The activity of PP i -ase in cell-free extracts of methanotrophs was considerably lower than in cell-free extracts of most of the methylobacteria studied and E. coli K12.

Functional Characterization of the Plastidic Phosphate Translocator Gene Family from the Thermo-Acidophilic Red Alga Galdieria sulphuraria Reveals Specific Adaptations of Primary Carbon Partitioning in Green Plants and Red Algae

In chloroplasts of green plants and algae, CO(2) is assimilated into triose-phosphates (TPs); a large part of these TPs is exported to the cytosol by a TP/phosphate translocator (TPT), whereas some is stored in the plastid as starch. Plastidial phosphate translocators have evolved from transport proteins of the host endomembrane system shortly after the origin of chloroplasts by endosymbiosis. The red microalga Galdieria sulphuraria shares three conserved putative orthologous transport proteins with the distantly related seed plants and green algae. However, red algae, in contrast to green plants, store starch in their cytosol, not inside plastids. Hence, due to the lack of a plastidic starch pool, a larger share of recently assimilated CO(2) needs to be exported to the cytosol. We thus hypothesized that red algal transporters have distinct substrate specificity in comparison to their green orthologs. This hypothesis was tested by expression of the red algal genes in yeast (Saccharomyces cerevisiae) and assessment of their substrate specificities and kinetic constants. Indeed, two of the three red algal phosphate translocator candidate orthologs have clearly distinct substrate specificities when compared to their green homologs. GsTPT (for G. sulphuraria TPT) displays very narrow substrate specificity and high affinity; in contrast to green plant TPTs, 3-phosphoglyceric acid is poorly transported and thus not able to serve as a TP/3-phosphoglyceric acid redox shuttle in vivo. Apparently, the specific features of red algal primary carbon metabolism promoted the evolution of a highly efficient export system with high affinities for its substrates. The low-affinity TPT of plants maintains TP levels sufficient for starch biosynthesis inside of chloroplasts, whereas the red algal TPT is optimized for efficient export of TP from the chloroplast.

Aminoacetone, a Putative Endogenous Source of Methylglyoxal, Causes Oxidative Stress and Death to Insulin-Producing RINm5f Cells

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4(+) ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O2(*-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 microM) and Fe(3+) ion (100 microM) addition to the cell cultures had no effect, whereas Cu(2+) (5.0-100 microM) significantly increased cell death. Supplementation of the AA- and Cu(2+)-containing culture medium with antioxidants, such as catalase (5.0 microM), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu(2+) ions (10-50 microM) relative to control cultures. This may imply higher activity of antioxidant enzymes in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) plus Cu(2+) (100 microM) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Metabolomic and Mass Isotopomer Analysis of Liver Gluconeogenesis and Citric Acid Cycle: II. HETEROGENEITY OF METABOLITE LABELING PATTERN

In this second of two companion articles, we compare the mass isotopomer distribution of metabolites of liver gluconeogenesis and citric acid cycle labeled from NaH(13)CO(3) or dimethyl [1,4-(13)C(2)]succinate. The mass isotopomer distribution of intermediates reveals the reversibility of the isocitrate dehydrogenase + aconitase reactions, even in the absence of a source of alpha-ketoglutarate. In addition, in many cases, a number of labeling incompatibilities were found as follows: (i) glucose versus triose phosphates and phosphoenolpyruvate; (ii) differences in the labeling ratios C-4/C-3 of glucose versus (glyceraldehyde 3-phosphate)/(dihydroxyacetone phosphate); and (iii) labeling of citric acid cycle intermediates in tissue versus effluent perfusate. Overall, our data show that gluconeogenic and citric acid cycle intermediates cannot be considered as sets of homogeneously labeled pools. This probably results from the zonation of hepatic metabolism and, in some cases, from differences in the labeling pattern of mitochondrial versus extramitochondrial metabolites. Our data have implications for the use of labeling patterns for the calculation of metabolic rates or fractional syntheses in liver, as well as for modeling liver intermediary metabolism.

Decreasing Intracellular Superoxide Corrects Defective Ischemia-induced New Vessel Formation in Diabetic Mice

Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1alpha by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1alpha modification by methylglyoxal reduced heterodimer formation and HIF1alpha binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes.

Use of expression analysis to dissect alterations in carbohydrate metabolism in wheat leaves during drought stress

Water deficit in plants causes a reduction in photosynthesis and high demands for osmolyte synthesis. To elucidate regulation of carbohydrate metabolic genes in wheat (Triticum aestivum) leaves during drought stress, we performed a systematic expression study using quantitative RT-PCR and cDNA microarray. These analyses revealed that expression levels of most genes encoding chloroplast enzymes involved in carbon fixation (Calvin cycle) were reduced in the leaves during prolonged drought stress. Transcript levels of highly expressed isoenzymes of hexokinase and fructokinase also decreased. Conversely, genes encoding cytoplasmic and vacuolar enzymes in the pathways leading to glucose, fructose and fructan production were up-regulated in the stressed leaves. Systematic expression analysis of an almost complete set of genes involved in conversion of triose phosphates to hexoses and hexose phosphorylation showed that isoenzymes of many enzymes were differentially regulated during drought stress. Correlation analysis indicated that the drought down-regulated Calvin cycle genes were coordinately regulated. This coordinated down-regulation extended to genes encoding major isoenzymes of chloroplast triosephosphate/phosphate translocator, cytoplasmic fructose-1,6-bisphosphate aldolase and fructose bisphosphatase. Highly correlated expression was also observed between drought up-regulated genes involved in sucrose synthesis and hydrolysis or fructan synthesis. These data dissect coordination in regulation of key enzyme genes involved in carbon fixation and accumulation of hexoses and fructans and provide an insight into molecular mechanisms at the transcript level underlying changes in carbohydrate metabolism in wheat adaptation to drought stress.

Regulation of plant cytosolic glyceraldehyde 3‐phosphate dehydrogenase isoforms by thiol modifications

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.

High Glucose Increases Angiopoietin-2 Transcription in Microvascular Endothelial Cells through Methylglyoxal Modification of mSin3A

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.

Stereospecific Proton Transfer by a Mobile Catalyst in Mammalian Fructose-1,6-bisphosphate Aldolase

Class I fructose-1,6-bisphosphate aldolases catalyze the interconversion between the enamine and iminium covalent enzymatic intermediates by stereospecific exchange of the pro(S) proton of the dihydroxyacetone-phosphate C3 carbon, an obligatory reaction step during substrate cleavage. To investigate the mechanism of stereospecific proton exchange, high resolution crystal structures of native and a mutant Lys(146) --> Met aldolase were solved in complex with dihydroxyacetone phosphate. The structural analysis revealed trapping of the enamine intermediate at Lys(229) in native aldolase. Mutation of conserved active site residue Lys(146) to Met drastically decreased activity and enabled trapping of the putative iminium intermediate in the crystal structure showing active site attachment by C-terminal residues 360-363. Attachment positions the conserved C-terminal Tyr(363) hydroxyl within 2.9A of the C3 carbon in the iminium in an orientation consistent with incipient re face proton transfer. We propose a catalytic mechanism by which the mobile C-terminal Tyr(363) is activated by the iminium phosphate via a structurally conserved water molecule to yield a transient phenate, whose developing negative charge is stabilized by a Lys(146) positive charge, and which abstracts the C3 pro(S) proton forming the enamine. An identical C-terminal binding mode observed in the presence of phosphate in the native structure corroborates Tyr(363) interaction with Lys(146) and is consistent with transient C terminus binding in the enamine. The absence of charge stabilization and of a mobile C-terminal catalyst explains the extraordinary stability of enamine intermediates in transaldolases.