Addition Of Malate Research Articles

Diesel exhaust particles alter adipose mitochondria bioenergetics

Diesel exhaust particles (DEP) constitute a significant quantity of air pollution that infiltrate the bloodstream to drive systemic pathologies. While some pathologies are obvious, such as COPD and heart failure, others are less apparent, such as insulin resistance, diabetes, and even obesity. In every case, a role for the receptor of advanced glycation end-products (RAGE) has been implicated. RAGE are cell surface receptors that are implicated in pathological processes, such as inflammation and diabetic complications. Despite the known connection between DEP and metabolic disorders, the effects of DEP exposure on adipose tissue mitochondrial function is unknown. To understand the potential role of RAGE signaling in the context of adipose tissue DEP exposure, mice were nasally exposed to either room air, nebulized diesel exhaust particles, or nebulized diesel exhaust particles + semi-synthetic glycosaminoglycan ethers (SAGEs, RAGE signaling inhibitors) every weekday for three weeks. Inguinal adipose tissue samples were obtained following exposure, and mitochondrial respiration rates were measured. In mice exposed to DEP or DEP + SAGE, mitochondrial respiration of inguinal adipose tissue was not significantly different compared to the room air control mice with the addition of glutamate and malate or ADP. However, respiration was significantly increased with the addition of succinate and FCCP in the inguinal adipose tissue from mice exposed to DEP. The results of this work indicate that DEP exposure elicits a negative effect on adipose mitochondrial respiration. This work was supported in part by funding from Levels Health (BTB) and the National Institutes of Health (NIH 1R15-HL152257, PRR and BTB). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Role of key enzymes in the production of docosahexaenoic acid (DHA) by Thraustochytrium sp. T01

Docosahexaenoic acid (DHA) is an essential dietary supplement that is highly coveted due to its benefits for human health. Extensive research has been conducted for the sustainable commercial production of DHA by various strains in thraustochytrid family due to the accumulation of higher lipid content in the cells. The current study is focused on improving DHA production by investigating various key enzymes like glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), and ATP-citrate lyase (ACL) involved in DHA production using Thraustochytrium sp. T01. The growth of this strain was compared in batch and fed-batch mode. The fed-batch yielded better Dry cell weight (40 g L−1), lipid (27.75 g L−1 or 693 mg g−1 of DCW), and DHA contents (11.10 g L−1 or 277 mg g−1 of DCW). G6PDH activity increased 4-fold during the glucose fed-batch, but ME and ACL did not increase significantly. Furthermore, a study was conducted to determine the effects of organic acids (pyruvate and malate) on key enzyme activities. The addition of pyruvate increased the lipid content by 1.35-fold, and ACL activity by 10-fold as compared with control (without added organic acids). Malate addition into the culture media increased DHA content 1.4-fold, and ME activity increased 14-fold compared with control.

Transcriptome analysis of malate-induced Schizochytrium sp. FJU-512 reveals a novel pathway for biosynthesis of docosahexaenoic acid with enhanced expression of genes responsible for acetyl-CoA and NADPH accumulation.

Schizochytrium is one of the few oleaginous microalgae that produce docosahexaenoic acid (DHA)-rich lipids. In this study, global changes in gene expression levels of Schizochytrium sp. FJU-512 cultured with malate in a 15 l-bioreactor was analyzed using comparative transcriptomics. The changes were found mainly in the genes involved in oxidative phosphorylation, β-oxidation, and pentose phosphate pathways. Consequently, the global changes in genes associated with the pathways could lead to an increase in the influx throughputs of pyruvate, branched-chain amino acids, fatty acids, and vitamin B6. Our transcriptome analysis indicated pyruvate dehydrogenase E2 component and acetolactate synthase I/II/III large subunit as major contributors to acetyl-CoA biosynthesis, whereas glucose-6-phosphate dehydrogenase was indicated as the major contributor to the biosynthesis of NADPH. An increase in DHA titer of up to 22% was achieved with the addition of malate to the fed-batch culture of Schizochytrium sp. FJU-512. This study provides an alternate method to enhance DHA production in Schizochytrium sp. FJU-512 through malate induced upregulation of genes responsible for acetyl-CoA and NADPH biosynthesis.

Effects of Matrix pH on Spontaneous Transient Depolarization and Reactive Oxygen Species Production in Mitochondria.

Reactive oxygen species (ROS) oxidize surrounding molecules and thus impair their functions. Since mitochondria are a major source of ROS, suppression of ROS overproduction in the mitochondria is important for cells. Spontaneous transient depolarization of individual mitochondria is a physiological phenomenon widely observed from plants to mammals. Mitochondrial uncoupling can reduce ROS production; therefore, it is conceivable that transient depolarization could reduce ROS production. However, transient depolarization has been observed with increased ROS production. Therefore, the exact contribution of transient depolarization to ROS production has not been elucidated. In this study, we examined how the spontaneous transient depolarization occurring in individual mitochondria affected ROS production. When the matrix pH increased after the addition of malate or exposure of the isolated mitochondria to a high-pH buffer, transient depolarization was stimulated. Similar stimulation by an increased matrix pH was also observed in the mitochondria in intact H9c2 cells. Modifying the mitochondrial membrane potential and matrix pH by adding K+ in the presence of valinomycin, a K+ ionophore, clarified that an increase in the matrix pH is a major cause of ROS generation. When we added ADP in the presence of oligomycin to suppress the transient depolarization without decreasing the matrix pH, we observed the suppression of mitochondrial respiration, increased matrix pH, and enhanced ROS production. Based on these results, we propose a model where spontaneous transient depolarization occurs during increased proton influx through proton channels opened by increased matrix pH, leading to the suppression of ROS production. This study improves our understanding of mitochondrial behavior.

Ca2+‐induced Complex I Inactivity: A Model for Early Mitochondrial Dysfunction Following Volumetric Muscle Loss Injury

Calcium (Ca2+) is a secondary messenger, responsible for the regulation of a plethora of metabolic pathways that must be tightly regulated to avoid cytotoxicity 1. During early stage volumetric muscle loss (VML) injuries, damaged muscle fibers are open to the extracellular environment until the myofiber can be sealed, a process that may take hours 2– 3. This allows for free Ca2+ exchange between intra‐ and extracellular environments to concentrations beyond those appropriate for cell homeostasis. The purpose of this study was to model the changes in mitochondrial metabolism of early VML injuries as a response to a drastic increase of intracellular Ca2+.C57BL6 mice underwent unilateral surgery to create a VML defect in the gastrocnemius muscle, as approved by the University of Georgia’s Institutional Animal Care and Use Committee. Animals were sacrificed via CO2 inhalation, 18 hrs. following surgery. Total [Ca2+] per kg of tissue ([Ca2+]TWM) was measured using a BAPTA complexation assay. Gastrocnemius muscles from naïve mice were dissected into individual fiber bundles, permeabilized, and washed in either normal isotonic or Ca2+‐supplemented medium. Mitochondrial metabolic function and mitochondrial membrane potential were measured through oxygen consumption and Safranin‐O fluorescence rates, respectively, in a High‐Resolution Fluoro‐respirometer.[Ca2+]TWM was elevated in VML‐injured tissue, as expected (n=3, p< 0.05). Acute Ca2+ exposure negatively affected Complex I (C‐I) activity of the electron transport chain. Specifically, C‐I activity was targeted by the addition of glutamate and malate (Glu‐Mal) and confirmed by the sequential addition of C‐I inhibitor Rotenone (Rot). In normal fibers, respiration increased after Glu‐Mal addition and subsequently decreased significantly following Rot, while Ca2+‐incubated fibers exhibited no change in oxygen consumption rate. Succinate (Succ) was used to rescue respiratory function by bypassing C‐I. Succ addition significantly increased oxygen consumption in both normal and Ca2+‐incubated fibers (n=3, p<0.01), confirming a C‐I dysfunction in Ca2+‐treated fibers. Safranin fluorescence is sensitive to changes in mitochondrial membrane potential, decreasing with complex activity and increasing upon inhibition. In normal fibers, Safranin signal decreased with Glu‐Mal and Succ additions and increased with Rot, as expected. However, Ca2+‐treated fibers were only sensitive to Succ treatment, further supporting C‐I dysfunction (n=3, p< 0.01).Herein, we show that an acute increase in intracellular Ca2+ negatively affects the process of oxidative phosphorylation within the mitochondria by disturbing the electron potential and inactivating Complex I. This alteration in membrane potential and decrease in complex activity may be the first step in a systematic shut down of mitochondrial metabolic function that leaves the muscle remaining after injury beyond the hope of effective repair.Support or Funding InformationFunded through the Department of Defense Neuromusculoskeletal Injuries Rehabilitation Research Award W81XWH‐18‐1‐0710 to SMG (UMinn) and JAC (UGA)A‐ Quantification of internal [Ca2+] of VML‐injured and control gastrocnemius muscles using BAPTA absorbance (n=3, p < 0.05). B‐ Oxygen consumption rates of control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3 in duplicate, p < 0.05). C‐ Safranin‐O signal fluctuation in control and Ca2+‐treated muscle fibers after Complex I activation, inhibition, and bypass, respectively (n=3, p< 0.05).Figure 1

P3483Diet governs metabolic and electrical properties of the atrial myocardium in mice

Abstract Background Obesity is associated with an increased risk of atrial fibrillation. This epidemiologic observation may not only be due to shared co-morbidities but also from direct impacts of metabolic disorders on myocardium Here, we examined the impact of high fat diet (HFD) induced obesity on atrial properties in mice. Methods Eight weeks old C57Bl/6J mice were subjected to 2 or 4 months of HFD (60% fat) or normal diet (ND, 4% fat). Rapid burst atrial pacing were delivered using a trans-esophageal probe to induced AF in anesthetized animals. Action potential (AP) were recorded in left atrial trabeculae using glass microelectrode technique. Potassium currents were recorded in isolated atrial myocytes using the whole cell or perforated patch clamp configurations. Metabolomic and lipidomic analysis were performed on whole left atria. Mitochondrial respiration was studied in situ in saponin-permeabilized atrial muscle fibres using a Clarke electrode and oxygen consumption was measured after successive addition of ADP (2 mM), malate (4 mM), palmitoyl-CoA and carnitine (pCoA-Ca) (100 μM and 2 mM), pyruvate (1 mM), glutamate (10 mM), succinate (15 mM), amytal (1 mM) and tetramethyl-paraphenylenediamine (TMPD)/ascorbate (0.5/0.5 mM). Results HFD mice showed a higher atrial vulnerability to AF as indicated by longer AF episodes compared to ND mice. APs were shorter in HFD versus ND mice (AP duration measured at 90% of repolarization: 47.9±2.4 msec in HFD vs 58.2±1.4 msec in ND, P<0.001 after 2 months of HFD) and more sensitive to the K ATP channel blocker, glibenclamide. In perforated but not whole cell, patch clamped atrial myocytes, the K-ATP component of the potassium current was enhanced in HFD mice (at +70 mV: 0.41±0.12 pA/pF in HFD vs 0.13±0.08 pA/pF in ND, P<0.05). Metabolomic analysis indicated an increased consumption of free fatty acid by the beta-oxidation and an accumulation of long chain fatty acid. Although the mitochondrial respiration measurements showed a trend towards a lower complex IV-driven respiration in HFD mice, no significant difference between ND and HFD was noted regarding complex I and complex II-driven respiration. However, the ability of fatty acids (pCoA-Car) to support mitochondrial respiration was higher in HFD Conclusions High fat diet induces a shift from carbon hydrate to a beta oxidation of the atrial myocardium metabolism together with an enhanced use of fatty acids by mitochondria. These metabolic changes could result in an enhanced activity of K-ATP channels and AP shortening that might contribute to AF vulnerability in this clinical setting. Acknowledgement/Funding ANR-10-IAHU-05

Effects of malate supplementation to the concentrate feed on performance, rumen fermentation and carcass yield of lambs fed forage at restricted and ad-libitum level

The effects of sodium and calcium salts of malic acid on growth performance, rumen pH, protozoa number, NH 3 - N level and volatile fatty acid (VFA) ratio and carcass yield in Akkaraman lambs fed alfalfa hay either restricted or ad-libitum were investigated. In a completely randomized design experiment, treatments were arranged at 2x2 factorial fashion: two forage levels (100 g/d or ad-libitum) and two malate levels (0 or 5 g/d), which were tested in 32 male lambs (3-4 months of age, 23.28±1.27 kg BW). Adaptation to feeding lasted 10 days; while sampling period lasted for 60 days. Growth performance, feed intake, slaughter and carcass weight were not affected by the treatments. A significant change was found in ruminal pH by forage feding level over time. At the beginning of the experiment the amount of acetic acid was increased by ad-libitum alfalfa hay consumption and malate addition but this effect was disappeared end of the trial. Rumen propionic acid, NH 3 -N concentration and protozoa number were not affected by the treatments but they changed by the sampling time. There was alfalfa hay level x malate supplementation x sampling time interaction effect on butyric acid concentration. In conclusion, malate addition did not improve growth rate and carcass yield of lambs but it affected ruminal acetic acid and butyric acid concentrations in lambs fed alfalfa ad-libitum.

Development and benefit evaluation of fermentation strategies for poly(malic acid) production from malt syrup by Aureobasidium melanogenum GXZ-6

Malt syrup, as a low-cost substrate without any pretreatment, was proved to be able to replace maltose for ploymalic acid (PMA) production by Aureobasidium melanogenum GXZ-6. The PMA titer of 55.53 ± 1.72 g/L was obtained by batch fermentation in a 10-L fermentor with addition of malate, citrate and sodium malonate. Then, a higher PMA titer of 124.07 ± 2.28 g/L was obtained in fed-batch fermentation, which increased by 123.43% than that from batch fermentation. Moreover, repeated-batch fermentation with three batches gave a PMA titer of 64.06 g/L on average with a higher yield of 0.81 g/g and productivity of 0.56 g/L·h. Fermentation process and economics analysis were performed by SuperPro Designer for a 2000 metric tons plant. Results showed that PMA production cost was as low as $ 1.716/kg by fed-batch fermentation, which provides an economical strategy for large-scale PMA production.

Monitoring of starter culture‐initiated liquid wheat and teff sourdough fermentations by selected ion flow tube‐mass spectrometry

Selected ion flow tube-mass spectrometry (SIFT-MS) is a direct-injection mass spectrometric technique that has been introduced recently into the field of food and flavor analysis. It also shows potential for use in the monitoring of food fermentations. Therefore, this study aimed at the online monitoring of different volatile compounds produced during starter culture-initiated liquid sourdough fermentations by SIFT-MS, for which a new workflow was developed. The online monitoring of the volatile sample compounds acetoin and ethyl acetate, diacetyl, and ethanol was made possible during the production of sourdoughs obtained through fermentation with several interesting strains belonging to the species Lactobacillus crustorum, Lactobacillus fermentum, Lactobacillus hilgardii, Lactobacillus nagelii, Lactobacillus sakei, and Gluconobacter oxydans. Acetoin and ethyl acetate could not be distinguished based solely on SIFT-MS data. Diacetyl production was monitored in the case of Lb. crustorum LMG 23699 as a starter culture strain, thereby making the distinction between those volatiles produced in sourdough without extra ingredients added or after the addition of citrate or malate. Starter culture-initiated liquid sourdough fermentations were monitored successfully. The volatile compound production of the different starter culture strains tested reflected differences in their metabolism and/or competitiveness in a sourdough matrix. © 2018 Society of Chemical Industry.

The mechanical properties of magnesium oxysulfate cement enhanced with 517 phase magnesium oxysulfate whiskers

A new magnesium oxysulfate (MOS) cement was prepared with the modification of sodium malate. The microscopic morphology of MOS cement was examined by scanning electron microscope (SEM). X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) was used to characterize the crystalline degree and molecular structure of MOS cement. The characterization results indicated that quantities of 517 phase magnesium oxysulfate whiskers were detected in the modified MOS cement. The flexural strength and water resistance of MOS cement were increased by the addition of sodium malate and the mechanism was also discussed according to the characterization results mentioned above.

GABA Production in Lactococcus lactis Is Enhanced by Arginine and Co-addition of Malate.

Lactococcus lactis NCDO 2118 was previously selected for its ability to decarboxylate glutamate to γ-aminobutyric acid (GABA), an interesting nutritional supplement able to improve mood and relaxation. Amino acid decarboxylation is generally considered as among the biochemical systems allowing lactic acid bacteria to counteracting acidic stress and obtaining metabolic energy. These strategies also include arginine deiminase pathway and malolactic fermentation but little is known about their possible interactions of with GABA production. In the present study, the effects of glutamate, arginine, and malate (i.e., the substrates of these acid-resistance pathways) on L. lactis NCDO 2118 growth and GABA production performances were analyzed. Both malate and arginine supplementation resulted in an efficient reduction of acidity and improvement of bacterial biomass compared to glutamate supplementation. Glutamate decarboxylation was limited to narrow environmental conditions (pH < 5.1) and physiological state (stationary phase). However, some conditions were able to improve GABA production or activate glutamate decarboxylation system even outside of this compass. Arginine clearly stimulated glutamate decarboxylation: the highest GABA production (8.6 mM) was observed in cultures supplemented with both arginine and glutamate. The simultaneous addition of arginine, malate, and glutamate enabled earlier GABA production (i.e., during exponential growth) at relatively high pH (6.5). As far as we know, no previous study has reported GABA production in such conditions. Although further studies are needed to understand the molecular basis of these phenomena, these results represent important keys suitable of application in GABA production processes.

Renal Fanconi Syndrome Is Caused by a Mistargeting-Based Mitochondriopathy.

We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity,mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.

Reconstruction and analysis of the genome-scale metabolic model of schizochytrium limacinum SR21 for docosahexaenoic acid production.

BackgroundSchizochytrium limacinum SR21 is a potential industrial strain for docosahexaenoic acid (DHA) production that contains more than 30–40 % DHA among its total fatty acids.MethodsTo resolve the DHA biosynthesis mechanism and improve DHA production at a systematic level, a genomescale metabolic model (GSMM), named iCY1170_DHA, which contains 1769 reactions, 1659 metabolites, and 1170 genes, was reconstructed.ResultsBased on genome annotation results and literature reports, a new DHA synthesis pathway based on a polyketide synthase (PKS) system was detected in S. limacinum. Similarly to conventional fatty acid synthesis, the biosynthesis of DHA via PKS requires abundant acetyl-CoA and NADPH. The in silico addition of malate and citrate led to increases of 24.5 % and 37.1 % in DHA production, respectively. Moreover, based on the results predicted by the model, six amino acids were shown to improve DHA production by experiment. Finally, 30 genes were identified as potential targets for DHA over-production using a Minimization of Metabolic Adjustment algorithm.ConclusionsThe reconstructed GSMM, iCY1170_DHA, could be used to elucidate the mechanism by which DHA is synthesized in S. limacinum and predict the requirements of abundant acetyl-CoA and NADPH for DHA production as well as the enhanced yields achieved via supplementation with six amino acids, malate, and citrate.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2042-y) contains supplementary material, which is available to authorized users.

The impact of ageing on adipose structure, function and vasculature in the B6D2F1 mouse: evidence of significant multisystem dysfunction.

The critical influence of the white adipose tissue (WAT) on metabolism is well-appreciated in obesity, but adipose tissue dysfunction as a mechanism underlying age-associated metabolic dysfunction requires elucidation. To explore this possibility, we assessed metabolism and measures of epididymal (e)WAT mitochondria and artery function in young (6.1 ± 0.4 months) and old (29.6 ± 0.2 months) B6D2F1 mice. There were no group differences in average daily oxygen consumption, fasted blood glucose or plasma free fatty acids, but fasted plasma insulin and the homeostatic model assessment of insulin resistance (HOMA-IR%) were higher in the old (∼50-85%, P < 0.05). Tissue mass (P < 0.05) and adipocyte area were lower (∼60%) (P < 0.01) and fibrosis was greater (sevenfold, P < 0.01) in eWAT with older age. The old also exhibited greater liver triglycerides (∼60%, P < 0.05). The mitochondrial respiratory oxygen flux after the addition of glutamate and malate (GM), adenosine diphosphate (d), succinate (S) and octanoyl carnitine (O) were one- to twofold higher in eWAT of old mice (P < 0.05). Despite no change in the respiratory control ratio, substrate control ratios of GMOd/GMd and GMOSd/GMd were ∼30-40% lower in old mice (P < 0.05) and were concomitant with increased nitrotyrosine (P < 0.05) and reduced expression of brown adipose markers (P < 0.05). Ageing reduced vascularity (∼50%, P < 0.01), angiogenic capacity (twofold, P < 0.05) and expression of vascular endothelial growth factor (∼50%, P < 0.05) in eWAT. Finally, endothelium-dependent dilation was lower (P < 0.01) in isolated arteries from eWAT arteries of the old mice. Thus, metabolic dysfunction with advancing age occurs in concert with dysfunction in the adipose tissue characterized by both mitochondrial and arterial dysfunction.

Design and construction of a non-natural malate to 1,2,4-butanetriol pathway creates possibility to produce 1,2,4-butanetriol from glucose.

1,2,4-butanetriol (BT) is an important bulk chemical mainly used for producing the superior energetic plasticizer (1,2,4-butanetriol trinitrate) in propellant and explosive formulations. BT is commercially produced by chemical synthesis from petroleum-based feedstocks; until recently a costly biosynthetic route from xylose or arabinose was reported. Here we designed a novel biosynthetic pathway for BT from malate, for the purpose of using glucose as an alternative and cheaper substrate in future. This biosynthetic pathway was achieved through six sequential enzymatic reactions. Following tests of several combinations of enzymes for the pathway, five enzymes including malate thiokinase, succinate-semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, 4-hydroxybutyrate CoA-transferase and bifunctional aldehyde/alcohol dehydrogenase were finally chosen. All enzyme genes were expressed on two compatible plasmids in E. coli, and their functions verified separately. Following assembly of two functional modules, BT was detected in the fermentation broth upon addition of malate, proving BT can be biosynthesized from malate. Furthermore, BT was detected in the fermentation using glucose as the sole carbon source, suggesting that such novel BT biosynthetic pathway has created the possibility for the production of BT from the cheaper substrate glucose.

Activity of the mitochondrial pyruvate dehydrogenase complex in plants is stimulated in the presence of malate

The effect of malate on the steady-state activity of the pea (Pisum sativum L.) and barley (Hordeum vulgare L.) leaf pyruvate dehydrogenase complex (PDC) has been studied in isolated mitochondria. The addition of malate was found to be stimulatory for the mitochondrial PDC, however there was no stimulation of chloroplast PDC. The stimulation was saturated below 1mM malate and was apparently related to а partially activated complex, which activity increased in the presence of malate by about twofold. Malate also reversed the reduction of PDC activity in the presence of glycine. Based on the obtained kinetic data, we suggest that the effect of malate is rather not a direct activation of PDC but involves the establishment of NAD-malate dehydrogenase equilibrium, decreasing concentration of NADH and relieving its inhibitory effect of PDC.

Phosphoenolpyruvate Carboxylase Identified as a Key Enzyme in Erythrocytic Plasmodium falciparum Carbon Metabolism

Phospoenolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C,15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery.

Malate and Fumarate Extend Lifespan in Caenorhabditis elegans

Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in the nematode C. elegans. Malate can be synthesized from fumarate by the enzyme fumarase and further oxidized to oxaloacetate by malate dehydrogenase with the accompanying reduction of NAD. Addition of fumarate also extended lifespan, but succinate addition did not, although all three intermediates activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced oxidative stress. The glyoxylate shunt, an anabolic pathway linked to lifespan extension in C. elegans, reversibly converts isocitrate and acetyl-CoA to succinate, malate, and CoA. The increased longevity provided by malate addition did not occur in fumarase (fum-1), glyoxylate shunt (gei-7), succinate dehydrogenase flavoprotein (sdha-2), or soluble fumarate reductase F48E8.3 RNAi knockdown worms. Therefore, to increase lifespan, malate must be first converted to fumarate, then fumarate must be reduced to succinate by soluble fumarate reductase and the mitochondrial electron transport chain complex II. Reduction of fumarate to succinate is coupled with the oxidation of FADH2 to FAD. Lifespan extension induced by malate depended upon the longevity regulators DAF-16 and SIR-2.1. Malate supplementation did not extend the lifespan of long-lived eat-2 mutant worms, a model of dietary restriction. Malate and fumarate addition increased oxygen consumption, but decreased ATP levels and mitochondrial membrane potential suggesting a mild uncoupling of oxidative phosphorylation. Malate also increased NADPH, NAD, and the NAD/NADH ratio. Fumarate reduction, glyoxylate shunt activity, and mild mitochondrial uncoupling likely contribute to the lifespan extension induced by malate and fumarate by increasing the amount of oxidized NAD and FAD cofactors.

Promoting Influence of Organic Carbon Source on Chromate Reduction by &lt;i&gt;Bacillus&lt;/i&gt; sp.

The effect of different organic carbon sources on Cr(Ⅵ) reduction by Bacillus sp. was investigated. Three organic compounds, malate, succinate and glucose were tested in Cr(Ⅵ) reduction experiments. The results demonstrated that three organic compounds all significantly enhanced Cr(Ⅵ) reduction. The enhancing degree of these three organic compounds was observed as malate&gt;glucose&gt;succinate, although the cell growth in succinate-added medium was similar to that in malate-added medium and was apparently higher than that in glucose-added medium. Addition of glucose resulted in decrease of pH and increase of redox potential, while addition of malate or succinate resulted in increase of pH and decrease of redox potential during Cr(Ⅵ) reduction. Cr(Ⅵ) was reduced by liquid culture metabolite when glucose was added in liquid medium, but not reduced by liquid culture metabolite when malate or succinate was applied in liquid medium for bacterial cultivation.

Skeletal muscle mitochondrial function: is it quality or quantity that makes the difference in insulin resistance?

), during heightened metabolicdemand (i.e. contraction) there is anessential requirement for rapid andsustained ATP production, a role fulfilledprimarilybymitochondria.Assuch,skeletalmuscle cells are densely populated withthese complex organelles.Withanincreasingprevalenceofinactivityand obesity, type 2 diabetes has become amajor public health concern. Given theircentral role in energy production, it isperhaps not surprising that mitochondrialdysfunction has been implicated inthe aetiology of skeletal muscle insulinresistance, a precursor of frank diabetes.Furthermore,interventionswhichstimulateskeletal muscle mitochondrial biogenesiscan improve insulin sensitivity in obeseand/or insulin resistant individuals. Whileit seems clear that a reduction inskeletal muscle mitochondrial