Cytosolic Malate Dehydrogenase Research Articles

The effect of electroaucpuncture for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced proteomic changes in the mouse striatum

Using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinson's disease mouse model, we investigated protein expression changes associated with the action of electroacupuncture (EA) in the mouse striatum. Twelve-week-old male C57BL/6 mice were injected intraperitoneally with 30 mg/kg of MPTP at 24-h intervals for 5 days, and the 100-Hz EA stimulation was performed at GB34 and GB39 once a day for 12 days consecutively from the first injection. With the EA, the MPTP-induced dopaminergic neuronal destruction was reduced. Of the 13 proteins that were differentially expressed between control and MPTP treated mice, cytosolic malate dehydrogenase, munc18-1, and hydroxyacylglutathione hydrolase, which were increased by MPTP, and cytochrome c oxidase subunit Vb, which was decreased by MPTP, were restored to the level of the saline group after EA treatment. These proteins are likely related to cellular metabolism. Altogether, we propose that the EA may exert neuroprotective effects in mice striatum through reducing MPTP-induced toxicity such as oxidative stress.

A Novel Decoy That Interrupts G93A-Superoxide Dismutase Gain of Interaction with Malate Dehydrogenase Improves Survival in an Amyotrophic Lateral Sclerosis Cell Model

Human G93A-superoxide dismutase-1 (G93AhSOD1) mutation causes amyotrophic lateral sclerosis (ALS) in rodents and humans. Recent observations indicate gain of interaction of G93AhSOD1 with cytosolic malate dehydrogenase (MDH1) and subsequent impairment in the malate aspartate shuttle which is vital to neurons. Using fluorescence resonance energy transfer (FRET), we screened an MDH1 derived peptide library for a decoy that would interrupt the G93AhSOD1-MDH1 interaction. A specific 23 amino acid blocker of this interaction was thus discovered, and interruption of interaction was confirmed by pull-down immunoprecipitation studies. A cell permeable 5-carboxytetramethylrhodamine derivative of the decoy peptide improved ATP content of motor neuron derived NSC-34 cells expressing G93AhSOD1 and enhanced cell survival under rotenone and low glucose challenges. Decoy agents capable of interrupting the gain of toxic interaction of G93AhSOD1 with MDH1 provide further evidence for the role of malate aspartate shuttle inhibition in G93AhSOD1 toxicity and a promising new route in ALS drug research.

Availability of neurotransmitter glutamate is diminished when β‐hydroxybutyrate replaces glucose in cultured neurons

Ketone bodies serve as alternative energy substrates for the brain in cases of low glucose availability such as during starvation or in patients treated with a ketogenic diet. The ketone bodies are metabolized via a distinct pathway confined to the mitochondria. We have compared metabolism of [2,4-(13)C]beta-hydroxybutyrate to that of [1,6-(13)C]glucose in cultured glutamatergic neurons and investigated the effect of neuronal activity focusing on the aspartate-glutamate homeostasis, an essential component of the excitatory activity in the brain. The amount of (13)C incorporation and cellular content was lower for glutamate and higher for aspartate in the presence of [2,4-(13)C]beta-hydroxybutyrate as opposed to [1,6-(13)C]glucose. Our results suggest that the change in aspartate-glutamate homeostasis is due to a decreased availability of NADH for cytosolic malate dehydrogenase and thus reduced malate-aspartate shuttle activity in neurons using beta-hydroxybutyrate. In the presence of glucose, the glutamate content decreased significantly upon activation of neurotransmitter release, whereas in the presence of only beta-hydroxybutyrate, no decrease in the glutamate content was observed. Thus, the fraction of the glutamate pool available for transmitter release was diminished when metabolizing beta-hydroxybutyrate, which is in line with the hypothesis of formation of transmitter glutamate via an obligatory involvement of the malate-aspartate shuttle.

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.

Metabolic Enzymes, Antioxidants, and Cytoskeletal Proteins Are Significantly Altered in Vastus Lateralis Muscle of K-Depleted Cadaveric Subjects

Molecular mechanisms underlying myopathy caused by prolonged potassium (K) depletion remain poorly understood. In the present study, we examined proteome profile of vastus lateralis muscle obtained from cadaveric subjects who had K depletion (KD) (muscle K<80 micromol/g wet weight) compared to those who had no KD (NKD) (muscle K>or=80 micromol/g wet weight) (n=6 per group). Muscle proteins were extracted, resolved by 2-DE, and visualized with CBB-R250 stain. Spot matching and intensity analysis revealed significant changes in levels of 11 (6 increased and 5 decreased) protein spots in the KD group. Q-TOF MS and MS/MS analyses identified these altered proteins as metabolic enzymes (aldehyde dehydrogenase 1A1, uridine diphosphoglucose pyrophosphorylase, enolase 1, cytosolic malate dehydrogenase, and carbonic anhydrase III), antioxidants (peroxiredoxin-3 isoform b), cytoskeletal proteins (slow-twitch skeletal troponin I and myosin light chain 2), and others. These altered proteins are involved in many cellular functions, including bioenergetics, acid-base regulation, oxidative stress response, and muscle contractility. Validation was done by Western blot analysis, which confirmed the increased level of peroxiredoxin-3 and decreased level of troponin-I in the KD muscle. Linear regression analysis also revealed a significant negative correlation between peroxiredoxin-3 level and muscle K content (r=-0.887; p<0.001), as well as a significant positive correlation between troponin-I level and muscle K content (r=0.618; p<0.05). Our results implicate the important roles these altered proteins play in the development of KD-associated myopathy.

Decreased levels of metabolic enzymes in pancreatic islets of patients with type 2 diabetes

Glucose-stimulated insulin secretion is defective in patients with type 2 diabetes. We sought to acquire new information about enzymes of glucose metabolism, with an emphasis on mitochondrial enzymes, by comparing pancreatic islets of type 2 diabetes patients with those of non-diabetic controls. Expression of genes encoding 13 metabolic enzymes was estimated with microarrays and activities of up to nine metabolic enzymes were measured. The activities of the mitochondrial enzymes, glycerol phosphate dehydrogenase, pyruvate carboxylase (PC) and succinyl-CoA:3-ketoacid-CoA transferase (SCOT) were decreased by 73%, 65% and 92%, respectively, in the diabetic compared with the non-diabetic islets. ATP citrate lyase, a cytosolic enzyme of the mitochondrial citrate pyruvate shuttle, was decreased 57%. Activities of propionyl-CoA carboxylase, NADP-isocitrate dehydrogenase, cytosolic malic enzyme, aspartate aminotransferase and malate dehydrogenase were not significantly different from those of the control. The low activities of PC and SCOT were confirmed with western blots, which showed that their protein levels were low. The correlation of relative mRNA signals with enzyme activities was good in four instances, moderate in four instances and poor in one instance. In diabetic islets, the mRNA signal of the islet cell-enriched transcription factor musculoaponeurotic fibrosarcoma oncogene homologue A, which regulates expression of islet genes, including the PC gene, was decreased to 54% of the control level. PC activity and protein levels in the non-diabetic islets were significantly lower than in islets from non-diabetic rodents. Low levels of certain islet metabolic enzymes, especially mitochondrial enzymes, are associated with human type 2 diabetes.

Cloning, expression and biochemical characterization of mitochondrial and cytosolic malate dehydrogenase from Phytophthora infestans

The genes of the mitochondrial and cytosolic malate dehydrogenase (mMDH and cMDH) of Phytophthora infestans were cloned and overexpressed in Escherichia coli as active enzymes. The catalytic properties of these proteins were determined: both enzymes have a similar specific activity. In addition, the natural mitochondrial isoenzyme was semi-purified from mycelia and its catalytic properties determined: the recombinant mitochondrial isoform behaved as the natural enzyme. A phylogenetic analysis indicated that mMDH, present in all stramenopiles studied, can be useful to study the relationships between these organisms. MDH with the conserved domain MDH_cytoplasmic_cytosolic is absent in some stramenopiles as well as in fungi. This enzyme seems to be less related within the stramenopile group. The Phytophthora cMDHs have an insertion of six amino acids that is also present in the stramenopile cMDHs studied, with the exception of Thalassiosira pseudonana cMDH, and is absent in other known eukaryotic cMDHs.

Direct evidence that genetic variation in glycerol-3-phosphate and malate dehydrogenase genes (Gpdh and Mdh1) affects adult ethanol tolerance in Drosophila melanogaster.

Many studies of alcohol adaptation in Drosophila melanogaster have focused on the Adh polymorphism, yet the metabolic elimination of alcohol should involve many enzymes and pathways. Here we evaluate the effects of glycerol-3-phosphate dehydrogenase (Gpdh) and cytosolic malate dehydrogenase (Mdh1) genotype activity on adult tolerance to ethanol. We have created a set of P-element-excision-derived Gpdh, Mdh1, and Adh alleles that generate a range of activity phenotypes from full to zero activity. Comparisons of paired Gpdh genotypes possessing 10 and 60% normal activity and 66 and 100% normal activity show significant effects where higher activity increases tolerance. Mdh1 null allele homozygotes show reductions in tolerance. We use piggyBac FLP-FRT site-specific recombination to create deletions and duplications of Gpdh. Duplications show an increase of 50% in activity and an increase of adult tolerance to ethanol exposure. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection related to adaptation to alcohols. Finally, we examine the interactions between activity genotypes for Gpdh, Mdh1, and Adh. We find no significant interlocus interactions. Observations on Mdh1 in both Gpdh and Adh backgrounds demonstrate significant increases in ethanol tolerance with partial reductions (50%) in cytosolic MDH activity. This observation strongly suggests the operation of pyruvate-malate and, in particular, pyruvate-citrate cycling in adaptation to alcohol exposure. We propose that an understanding of the evolution of tolerance to alcohols will require a system-level approach, rather than a focus on single enzymes.

Temperature adaptation of cytosolic malate dehydrogenases of limpets(genusLottia): differences in stability and function due to minor changes in sequence correlate with biogeographic and vertical distributions

We characterized functional and structural properties of cytoplasmic malate dehydrogenases (cMDHs) from six limpets of the genus Lottia that have different vertical and latitudinal distributions. Particular attention was given to the cryptic species pair Lottia digitalis (northern occurring) and L. austrodigitalis (southern occurring) because of recent contraction in the southern range of L. digitalis and a northward range extension of L. austrodigitalis. As an index of adaptation of function, we measured the effects of temperature on the apparent Michaelis-Menten constant (K(m)) of the cofactor NADH (K(m)(NADH)). K(m)(NADH) values of cMDHs from the mid- to high-intertidal, low-latitude species L. scabra and L. gigantea were less sensitive to high temperature than those of cMDHs from the low- and mid-intertidal, high-latitude species L. scutum and L. pelta. cMDH of L. digitalis was more sensitive to high temperatures than the cMDH ortholog of L. austrodigitalis. Thermal stability (rate of loss of activity at 42.5 degrees C) showed a similar pattern of interspecific variation. Comparison of the deduced amino acid sequences showed that interspecific differences ranged from one to as many as 17 residues. Differences in K(m)(NADH) and thermal stability between orthologs of L. digitalis and L. austrodigitalis result from a single amino acid substitution. At position 291, the glycine residue in cMDH of L. digitalis is replaced by a serine in cMDH of L. austrodigitalis, a change that favors additional hydrogen bonding and reduced conformational entropy. This difference between closely related congeners demonstrates the role of minor alterations in protein sequence in temperature adaptation and suggests that such variation is important in governing shifts in biogeographic range in response to climate change.

Localization of the Nucleic Acid Channel Regulatory Subunit, Cytosolic Malate Dehydrogenase

NACh is a nucleic acid-conducting channel found in apical membrane of rat kidney proximal tubules. It is a heteromultimeric complex consisting of at least two proteins: a 45-kDa pore-forming subunit and a 36-kDa regulatory subunit. The regulatory subunit confers ion selectivity and influences gating kinetics. The regulatory subunit has been identified as cytosolic malate dehydrogenase (cMDH). cMDH is described in the literature as a soluble protein that is not associated with plasma membrane. Yet a role for cMDH as the regulatory subunit of NACh requires that it be present at the plasma membrane. To resolve this conflict, studies were initiated to determine whether cMDH could be found at the plasma membrane. Before performing localization studies, a suitable model system that expressed NACh was identified. A channel was identified in LLC-PK(1) cells, a line derived from pig proximal tubule, that is selective for nucleic acid and has a conductance of approximately 10 pS. It exhibits dose-dependent blockade by heparan sulfate or L-malate. These characteristics are similar to what has been reported for NACh from rat kidney and indicate that NACh is present in LLC-PK(1) cells. LLC-PK(1) cells were therefore used as a model system for immunolocalization of cMDH. Both immunofluorescence and immunoelectron microscopy demonstrated cMDH at the plasma membrane of LLC-PK(1) cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.

Regional differences in porcine adipocytes isolated from skeletal muscle and adipose tissues as identified by a proteomic approach

The content and distribution of body lipids are of special interest for production efficiency and meat quality in the farm animal industry. Triglycerides represent the most variable fraction of tissue lipids, and are mainly stored in adipocytes. Although several studies have reported regional differences in the expression of genes and their products in adipocytes from various species, the characteristics of i.m. adipocytes remain poorly described. To evaluate adipocyte features according to muscle and other fat locations, adipocyte proteins were isolated from trapezius skeletal muscle, and intermuscular, s.c., or perirenal adipose tissues from 6 female pigs (80 d of age). Protein extracts were labeled and analyzed by 2-dimensional, fluorescent, differential gel electrophoresis. The comparisons revealed that 149 spots were always differentially expressed (P < 0.05, ratio exceeding |2|-fold difference) between i.m. adipocytes and the fat cells derived from the 3 other adipose locations. The proteins that were downregulated in i.m. fat cells belonged to various metabolic pathways, such as lipogenesis (cytosolic malate dehydrogenase and isocitrate dehydrogenase, P < 0.01), glycolysis (enolases and aldolase, P </= 0.01), lipolysis (perilipin, P < 0.01), fatty acid oxidation (long-chain fatty-acyl CoA dehydrogenase, P < 0.01), and energy transfer (catalase, voltage-dependent anion channel 1, and electron-transfer flavoprotein, P < 0.05). In contrast, both prohibitin-1 and cell division cycle 42 homolog, with possible roles in cell growth, were up-regulated (P < 0.05) in i.m. adipocytes compared with other fat cells. Fewer differences were observed when adipocytes isolated from s.c., perirenal, and intermuscular fat tissues were compared, with a maximum of 17 spots differing significantly in abundance between perirenal and s.c. adipose tissues. The findings that proteins involved in both anabolic and energy-yielding catabolic pathways are downregulated in i.m. adipocytes compared with s.c., visceral, or intermuscular adipocytes, suggest that the metabolic activity of i.m. adipocytes is low. Thus, triggering adipogenesis rather than cell metabolism per se might be a valuable strategy to control lipid deposition in pig skeletal muscles.

Gene clone, expression and enzyme activity assay of a cytosolic malate dehydrogenase from apple fruits

Malate dehydrogenase (MDH) ubiquitously exists in animals, plants and microoganisms, and catalyzes the interconversion from oxaloacetate to malate. Cytosolic NAD-dependent MDH gene (cyMDH) encodes a key enzyme crucial for malic acid synthesis in the cytosol which has not been extensively characterized in plants. In this study, a full-length cDNA of cyMDH was isolated from apple fruits with RT-PCR as well as 3′ and 5′ rapid amplification of cDNA ends, and designated as Mal-cyMDH (GenBank accession No. DQ221207). It contained a 996-bp ORF and its sequence analysis shows a high similarity to other plant cyMDHs. Phylogenetic analysis indicated that almost all the cyMDHs could be clustered into the same group and it was likely to represent the original MDH. A roughly 37-kDa fused protein was obtained by the recombinant prokaryotic expression and its enzyme activity assay showed that it mainly catalyzed oxaloacetate to malate. It was also discovered that the enzyme activity of cyMDH exhibited remarkable difference between the high-and low-acid apple germplasm.

Gain of interaction of ALS-linked G93A superoxide dismutase with cytosolic malate dehydrogenase

Protein interactions of the Amyotrophic Lateral Sclerosis (ALS)-linked copper–zinc superoxide dismutase (hSOD1) G93A mutation were studied using a fluorescence resonance energy transfer (FRET) based screening system. The FRET results confirmed by pull-down immunoprecipitation indicated “gain-of-interaction” of the G93A-hSOD1 mutant with cytosolic malate dehydrogenase (cytMDH)—a key enzyme in the malate–aspartate shuttle which is vital to neurons. Furthermore, cytMDH mRNA expression was upregulated in G93A-hSOD1 expressing cells but endogenous cytMDH enzymatic activity was not enhanced, not even with exogenously added-on enzyme. Consistent with inhibition of the malate–aspartate shuttle, G93A-hSOD1 had lower malate and higher lactate levels compared to non-induced or Wild-Type-hSOD1 expressing cells. Mitochondrial NADH/NAD+ ratio is also elevated. Malate–aspartate shuttle dysfunction may explain the damage to neurons and the vulnerability to impairments of glycolytic pathways in ALS and provide a new target for the development of potential therapies.

Localisation of gluconeogenesis and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of the TCA cycle in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii displays some unusual localisations of carbohydrate converting enzymes, which is due to the presence of a vestigial, non-photosynthetic plastid, referred to as the apicoplast. It was recently demonstrated that the single pyruvate dehydrogenase complex (PDH) in T. gondii is exclusively localised inside the apicoplast but absent in the mitochondrion. This raises the question about expression, localisation and function of enzymes for the tricarboxylic acid (TCA)-cycle, which normally depends on PDH generated acetyl-CoA. Based on the expression and localisation of epitope-tagged fusion proteins, we show that all analysed TCA cycle enzymes are localised in the mitochondrion, including both isoforms of malate dehydrogenase. The absence of a cytosolic malate dehydrogenase suggests that a typical malate-aspartate shuttle for transfer of reduction equivalents is missing in T. gondii. We also localised various enzymes which catalyse the irreversible steps in gluconeogenesis to a cellular compartment and examined mRNA expression levels for gluconeogenesis and TCA cycle genes between tachyzoites and in vitro bradyzoites. In order to get functional information on the TCA cycle for the parasite energy metabolism, we created a conditional knock-out mutant for the succinyl-CoA synthetase. Disruption of the sixth step in the TCA cycle should leave the biosynthetic parts of the cycle intact, but prevent FADH2 production. The succinyl-CoA synthetase depletion mutant displayed a 30% reduction in growth rate, which could be restored by supplementation with 2μM succinate in the tissue culture medium. The mitochondrial membrane potential in these parasites was found to be unaltered. The lack of a more severe phenotype suggests that a functional TCA cycle is not essential for T. gondii replication and for maintenance of the mitochondrial membrane potential.

Dehydroepiandrosterone supplement increases malate dehydrogenase activity and decreases NADPH-dependent antioxidant enzyme activity in rat hepatocellular carcinogenesis

Beneficial effects of dehydroepiandrosterone (DHEA) supplement on age-associated chronic diseases such as cancer, cardiovascular disease, insulin resistance and diabetes, have been reported. However, its mechanism of action in hepatocellular carcinoma in vivo has not been investigated in detail. We have previously shown that during hepatocellular carcinogenesis, DHEA treatment decreases formation of preneoplastic glutathione S-transferase placental form-positive foci in the liver and has antioxidant effects. Here we aimed to determine the mechanism of actions of DHEA, in comparison to vitamin E, in a chemically-induced hepatocellular carcinoma model in rats. Sprague-Dawley rats were administered with control diet without a carcinogen, diets with 1.5% vitamin E, 0.5% DHEA and both of the compounds with a carcinogen for 6 weeks. The doses were previously reported to have anti-cancer effects in animals without known toxicities. With DHEA treatment, cytosolic malate dehydrogenase activities were significantly increased by ~5 fold and glucose 6-phosphate dehydrogenase activities were decreased by ~25% compared to carcinogen treated group. Activities of Se-glutathione peroxidase in the cytotol was decreased significantly with DHEA treatment, confirming its antioxidative effect. However, liver microsomal cytochrome P-450 content and NADPH-dependent cytochrome P-450 reductase activities were not altered with DHEA treatment. Vitamin E treatment decreased cytosolic Se-glutathione peroxidase activities in accordance with our previous reports. However, vitamin E did not alter glucose 6-phosphate dehydrogenase or malate dehydrogenase activities. Our results suggest that DHEA may have decreased tumor nodule formation and reduced lipid peroxidation as previously reported, possibly by increasing the production of NADPH, a reducing equivalent for NADPH-dependent antioxidant enzymes. DHEA treatment tended to reduce glucose 6-phosphate dehydrogenase activities, which may have resulted in limited supply for de novo synthesis of DNA via inhibiting the hexose monophophaste pathway. Although both DHEA and vitamin E effectively reduced preneoplastic foci in this model, they seemed to function in different mechanisms. In conclusion, DHEA may be used to reduce hepatocellular carcinoma growth by targeting NADPH synthesis, cell proliferation and anti-oxidant enzyme activities during tumor growth.

Novel functions of the α-ketoglutarate dehydrogenase complex may mediate diverse oxidant-induced changes in mitochondrial enzymes associated with Alzheimer's disease

Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of α-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity. The present experiments tested whether both changes could be caused by the common oxidant H 2O 2 and to probe the mechanism underlying these changes. Since the response to H 2O 2 is modified by the level of the E2k subunit of KGDHC, the interaction of MDH and KGDHC was studied in cells with varying levels of E2k. In cells with only 23% of normal E2k protein levels, one-hour treatment with H 2O 2 decreased KGDHC and increased MDH activity as well as the mRNA level for both cytosolic and mitochondrial MDH. The increase in MDH did not occur in cells with 100% or 46% of normal E2k. Longer treatments with H 2O 2 inhibited the activity of both enzymes. Glutathione is a major regulator of cellular redox state and can modify enzyme activities. H 2O 2 converts reduced glutathione (GSH) to oxidized glutathione (GSSG), which reacts with protein thiols. Treatment of purified KGDHC with GSSG leads to glutathionylation of all three KGDHC subunits. Thus, cellular glutathione level was manipulated by two means to determine the effect on KGDHC and MDH activities. Both buthionine sulfoximine (BSO), which inhibits glutathione synthesis without altering redox state, and H 2O 2 diminished glutathione to a similar level after 24 h. However, H 2O 2, but not BSO, reduced KGDHC and MDH activities, and the reduction was greater in the E2k-23 line. These findings suggest that the E2k may mediate diverse responses of KGDHC and MDH to oxidants. In addition, the differential response of activities to BSO and H 2O 2 together with the in vitro interaction of KGDHC with GSSG suggests that glutathionylation is one possible mechanism underlying oxidative stress-induced inhibition of the TCA cycle enzymes.

Late onset of dietary restriction reverses age-related decline of malate–aspartate shuttle enzymes in the liver and kidney of mice

Dietary restriction (DR) influences several physiological processes, retards the incidences and severity of various age-related diseases and extends lifespan of various animal species. The effect of DR on the activities of malate-aspartate shuttle enzymes, viz. cytosolic and mitochondrial aspartate aminotransferase (c- and m-AsAT) and malate dehydrogenase (c- and m-MDH) was investigated in the liver and kidney of adult (5-months) and old (21-months) male mice. The results show that the activity (U/mg protein) of both c- and m-MDH and AsAT is decreased significantly in the liver and kidney of old mice compared to adult ones. However, DR in old mice reverses significantly the enzyme activities to a level closer to adult animals. Polyacrylamide gel electrophoresis (PAGE) and specific staining of c-AsAT, one of the selected isoenzymes of the shuttle, showed a similar pattern of activity expression as observed by activity measurements in both the tissues studied. Slot blot analysis of c-AsAT confirmed the lower protein content of this isoenzyme in old mice compared to adult ones and a higher level in old-dietary restricted mice. Thus, our results suggest that the late onset of DR in older mice reverses decline in malate-aspartate shuttle enzymes and that it may allow a better metabolic regulation in older animals.

Changes in metabolite, energy metabolism related enzyme activities and peripheral blood mononuclear cell (PBMC) Populations in beef heifers with two differing liveweight change profiles in New Zealand

Metabolite and immunoreactive insulin (IRI) concentrations, energy metabolism related enzymes activities and peripheral blood mononuclear cell (PBMC) populations were measured in blood of pregnant Angus heifers with differing liveweight change profiles (gaining or losing), in New Zealand to investigate the meanings of those parameters in the restricted feeding beef heifers. Beef heifers losing liveweight (-412 g/day) showed significantly lower concentrations of plasma IRI, and higher concentrations of plasma free fatty acid (FFA) than heifers gaining liveweight (483 g/day). The cytosolic and mitochondrial malate dehydrogenase (MDH) activities and MDH/lactate dehydrogenase (M/L) ratio in leukocytes of the liveweight losing heifers were significantly higher than those the liveweight gaining heifers. Percentages of cluster of differentiation (CD) 3 positive cells and natural killer (NK) cells in PBMC decreased significantly in the liveweight losing heifers compared to those in the liveweight gaining heifers. Plasma IRI and FFA concentrations, leukocyte cytosolic and mitochondrial MDH activities and CD3 positive and NK cell populations may be useful markers to evaluate metabolic conditions and immunity in the restricted feeding beef heifers.

Lactate and malate dehydrogenase in the fan-shell associated shrimp, Pontonia pinnophylax (Otto): Effects of temperature and urea

In Pontonia pinnophylax (Otto), a crustacean decapod inhabiting the mantle cavity of Pinna nobilis L. (Bivalvia: Pteriomorpha), the lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, and their electrophoretic patterns, were compared in relation to heat and urea inactivation. Activity was higher in LDH than in MDH, and the electrophoretic patterns showed a predominance of LDH-A4 and the presence of both mitochondrial and cytosolic MDH. Heat incubation reduced both enzymatic activities, but more MDH. Also all isozymes showed different heat sensitivity, with anodic forms more heat-resistant than cathodic ones, either in LDH as in MDH. Urea treatment caused also a higher inactivation of the most cathodic isozymes, but MDH appeared more resistant than LDH at 2 M urea. The high polymorphism of these enzymes suggests an adaptation of Pontonia metabolism to hypoxic conditions; moreover, the different isozyme stability grade should be functional to contrast environmental variability.

Effects of L-malate on physical stamina and activities of enzymes related to the malate-aspartate shuttle in liver of mice

L-malate, a tricarboxylic acid cycle (TCA) intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production and may be involved in the beneficial effects of improving physical stamina. In the present study, we investigated the effects of L-malate on the performance of forced swimming time and blood biochemical parameters related to fatigue - blood urea nitrogen (BUN), glucose (Glc), creatine kinase (CK),total protein (TP) and lactic acid (LA). To investigate the effects of L-malate on the malate-aspartate shuttle and energy metabolism in mice, the activities of enzymes related to the malate-aspartate shuttle were measured. L-malate was orally administered to mice continuously for 30 days using a feeding atraumatic needle. The swimming time was increased by 26.1 % and 28.5 %, respectively, in the 0.210 g/kg and 0.630 g/kg L-malate-treated group compared with the control group. There were no differences in the concentrations of Glc, BUN and TP between the L-malate-treated groups and the control groups. However, the levels of CK were significantly decreased in the L-malate-treated groups. The results predict a potential benefit of L-malate for improving physical stamina and minimizing muscle damage during swimming exercise. The activities of cytosolic and mitochondrial malate dehydrogenase were significantly elevated in the L-malate-treated group compared with the control group. These enzymatic activities may be useful indicators for evaluating changes affecting the malate-aspartate shuttle and energy metabolism in the liver of mice.