L-lactate metabolism in potato tuber mitochondria

We investigated the metabolism of L-lactate in mitochondria isolated from potato tubers grown and saved after harvest in the absence of any chemical agents. Immunologic analysis by western blot using goat polyclonal anti-lactate dehydrogenase showed the existence of a mitochondrial lactate dehydrogenase, the activity of which could be measured photometrically only in mitochondria solubilized with Triton X-100. The addition of L-lactate to potato tuber mitochondria caused: (a) a minor reduction of intramitochondrial pyridine nucleotides, whose measured rate of change increased in the presence of the inhibitor of the alternative oxidase salicyl hydroxamic acid; (b) oxygen consumption not stimulated by ADP, but inhibited by salicyl hydroxamic acid; and (c) activation of the alternative oxidase as polarographically monitored in a manner prevented by oxamate, an L-lactate dehydrogenase inhibitor. Potato tuber mitochondria were shown to swell in isosmotic solutions of ammonium L-lactate in a stereospecific manner, thus showing that L-lactate enters mitochondria by a proton-compensated process. Externally added L-lactate caused the appearance of pyruvate outside mitochondria, thus contributing to the oxidation of extramitochondrial NADH. The rate of pyruvate efflux showed a sigmoidal dependence on L-lactate concentration and was inhibited by phenylsuccinate. Hence, potato tuber mitochondria possess a non-energy-competent L-lactate/pyruvate shuttle. We maintain, therefore, that mitochondrial metabolism of L-lactate plays a previously unsuspected role in the response of potato to hypoxic stress.     

Transport and metabolism of L-lactate occur in mitochondria from cerebellar granule cells and are modified in cells undergoing low potassium dependent apoptosis

Having confirmed that externally added L-lactate can enter cerebellar granule cells, we investigated whether and how L-lactate is metabolized by mitochondria from these cells under normal or apoptotic conditions. (1) L-lactate enters mitochondria, perhaps via an L-lactate/H+ symporter, and is oxidized in a manner stimulated by ADP. The existence of an L-lactate dehydrogenase, located in the inner mitochondrial compartment, was shown by immunological analysis. Neither the protein level nor the Km and Vmax values changed en route to apoptosis. (2) In both normal and apoptotic cell homogenates, externally added L-lactate caused reduction of the intramitochondrial pyridine cofactors, inhibited by phenylsuccinate. This process mirrored L-lactate uptake by mitochondria and occurred with a hyperbolic dependence on L-lactate concentrations. Pyruvate appeared outside mitochondria as a result of external addition of L-lactate. The rate of the process depended on L-lactate concentration and showed saturation characteristics. This shows the occurrence of an intracellular L-lactate/pyruvate shuttle, whose activity was limited by the putative L-lactate/pyruvate antiporter. Both the carriers were different from the monocarboxylate carrier. (3) L-lactate transport changed en route to apoptosis. Uptake increased in the early phase of apoptosis, but decreased in the late phase with characteristics of a non-competitive like inhibition. In contrast, the putative L-lactate/pyruvate antiport decreased en route to apoptosis with characteristics of a competitive like inhibition in early apoptosis, and a mixed non-competitive like inhibition in late apoptosis.     

Synthesis and localization of L-lactate oxidase in yeasts Yarrowia lipolytica

Conditions for L-lactate oxidase synthesis by the yeast Yarrowia lipolytica were investigated. The enzyme was found to be synthesized during growth on L-lactate in the exponential growth phase. L-lactate oxidase synthesis was also observed on glucose after adaptation to stress conditions (oxidative or thermal stress) during the stationary growth phase after glucose consumption. The cells grown on L-lactate exhibited high levels of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase), which exceeded those of glucose-grown cells. Ultrastructurally, L-lactate-grown cells and the cells grown on glucose and adapted to various stress conditions were also found to be similar, with increased mitochondria, elevated number and size of peroxisomes, and formation of lipid and polyphosphate inclusions. In order to determine the intracellular localization of L-lactate oxidase, the cells were disintegrated by the lytic enzyme complex from Helix pomatia. Centrifugation of the homogenate in Percoll gradient resulted in the isolation of purified fractions of the native mitochondria and peroxisomes. L-lactate oxidase was shown to be localized in peroxisomes.     

Localization of L-lactate dehydrogenase in mitochondria

Relatively small but persistent amounts of L-lactate dehydrogenase (LDH) activity were found in mitochondrial preparations isolated from liver of the rat. Using a variety of cytosolic markers, it was found that essentially no cytosolic contamination was present. Respiratory velocities and respiratory control with L-lactate were somewhat lower than with glutamate, but equal or superior to those with pyruvate. Agarose gel electrophoresis showed LDH isoenzymes in mitochondria similar to that in corresponding cytosol. Subtilisin BPN', a bacterial protease, was incubated with intact mitochondria and enzyme activities were measured. Following mitochondrial disruption, the proteolytic treatment was repeated. Digitonin was also used in the fractionation of mitochondria. These techniques helped to determine the location of the LDH in the mitochondria as being mainly in the outer membrane and periplasmic space.     

A re-evaluation of the role of mitochondrial pyruvate transport in the hormonal control of rat liver mitochondrial pyruvate metabolism.

The inhibitor of mitochondrial pyruvate transport alpha-cyano-beta-(1-phenylindol-3-yl)-acrylate was used to inhibit progressively pyruvate carboxylation by liver mitochondria from control and glucagon-treated rats. The data showed that, contrary to our previous conclusions [Halestrap (1978) Biochem. J. 172, 389-398], pyruvate transport could not regulate metabolism under these conditions. This was confirmed by measuring the intramitochondrial pyruvate concentration, which almost equilibrated with the extramitochondrial pyruvate concentration in control mitochondria, but was significantly decreased in mitochondria from glucagon-treated rats, where rates of pyruvate metabolism were elevated. Computer-simulation studies explain how this is compatible with linear Dixon plots of the inhibition of pyruvate metabolism by alpha-cyano-4-hydroxycinnamate. Parallel measurements of the mitochondrial membrane potential by using [3H]triphenylmethylphosphonium ions showed that it was elevated by about 3 mV after pretreatment of rats with both glucagon and phenylephrine. There was no significant change in the transmembrane pH gradient. It is shown that the increase in pyruvate metabolism can be explained by a stimulation of the respiratory chain, producing an elevation in the protonmotive force and a consequent rise in the intramitochondrial ATP/ADP ratio, which in turn increases pyruvate carboxylase activity. Mild inhibition of the respiratory chain with Amytal reversed the effects of hormone treatment on mitochondrial pyruvate metabolism and ATP concentrations, but not on citrulline synthesis. The significance of these observations for the hormonal regulation of gluconeogenesis from L-lactate in vivo is discussed.     

The effect of various types of portocaval anastomoses and hepatic artery ligation on the metabolism of hepatic mitochondria in rats

The results of investigations are presented concerning the disorders of metabolism of mitochondria isolated from the liver of rats after various operations on the portal vein and hepatic artery. The mitochondria were studied after end-to-side portocaval anastomosis (PCA), end-to-side anastomosis of the mesenteric vein to the inferior caval vein (M-PCA) and after hepatic artery ligation. The investigations were carried out 3.5 months after these operations. The obtained results showed that the greatest and most significant statistically dissociation of oxidative phosphorylation and reduction of oxygen uptake developed in the mitochondria isolated from rats after M-PCA anastomosis. Simultaneously conducted investigations of citrulline production by suspension of mitochondria failed to demonstrate any changes in the urea cycle.     

The cytochrome c oxidase and its mitochondrial function in the whiteleg shrimp Litopenaeus vannamei during hypoxia

Cytochrome c oxidase (COX), which is located in the inner membrane of mitochondria, is a key constituent of the electron transport chain that catalyzes the reduction of oxygen. The Pacific whiteleg shrimp Litopenaeus vannamei is constantly exposed to hypoxic conditions, which affects both the central metabolism and the mitochondrial function. The purpose of this study was to isolate shrimp mitochondria, identify the COX complex and to evaluate the effect of hypoxia on the shrimp mitochondrial function and in the COX activity. A 190 kDa protein was identified as COX by immunodetection techniques. The effect of hypoxia was confirmed by an increase in the shrimp plasma L-lactate concentration. COX activity, mitochondrial oxygen uptake and protein content were reduced under hypoxic conditions, and gradually restored as hypoxia continued, this suggests an adaptive mitochondrial response and a highly effective COX enzyme. Both mitochondrial oxygen uptake and COX activity were completely inhibited by KCN and sodium azide, suggesting that COX is the unique oxidase in L. vannamei mitochondria.     

Improvement of L-lactate production by CYB2 gene disruption in a recombinant Saccharomyces cerevisiae strain under low pH condition

Using a DNA microarray, we found that expression of the genes related to lactate metabolism was upregulated in a lactate-producing recombinant Saccharomyces cerevisiae strain. Disruption of the CYB2 gene encoding L-lactate dehydrogenase improved the L-lactate production by S. cerevisiae under low pH condition.     

L-lactate oxidation by skeletal muscle mitochondria

1. Mitochondria isolated from rat skeletal muscle possess lactate dehydrogenase which is involved in direct oxidation of L-lactate in the presence of external NAD. 2. L-lactate oxidation can be stimulated in a reversible manner by ADP. 3. Mitochondrial lactate oxidation is sensitive to oxamate-inhibitor of LDH, alpha-cyano-3-hydroxy-cinnamate-pyruvate translocase inhibitor and respiratory chain inhibitors (rotenone, antimycin A, KCN). 4. In the same conditions the mitochondria did not oxidize pyruvate in the absence of malate, whereas, oxidize pyruvate plus external NADH in an uncoupling manner.     

Glyceraldehyde metabolism in human erythrocytes in comparison with that of glucose and dihydroxyacetone

Metabolism of D-glyceraldehyde in human erythrocytes in comparison with that of glucose and dihydroxyacetone was studied. Both trioses were metabolized to produce L-lactate at rates comparable to that of L-lactate formation from glucose. Almost complete inactivation of glyceraldehyde-3-phosphate dehydrogenase by treatment of cells with iodoacetate resulted in a 95% decrease in L-lactate formation from the ketotriose as well as from glucose, whereas L-lactate formation from the aldotriose was only partially reduced (60%). D-Lactate was produced faster from either the aldotriose or the ketotriose than from glucose, but the ability of the two trioses to produce D-lactate was far lower than that to produce L-lactate. Almost complete inhibition of aldehyde dehydrogenase by disulfiram and of both aldose reductase and aldehyde reductase II by sorbinil, had no effect on L-lactate formation from D-glyceraldehyde. The present study suggests that D-glyceraldehyde is metabolized via two or more pathways including the glycolytic pathway after its phosphorylation by triokinase, and that neither oxidation to D-glyceric acid nor reduction to glycerol is a prerequisite for D-glyceraldehyde metabolism.     

Kinetics, molecular basis, and differentiation of L-lactate transport in spermatogenic cells

Round spermatid energy metabolism is closely dependent on the presence of L-lactate in the external medium. This L-lactate has been proposed to be supplied by Sertoli cells in the seminiferous tubules. L-Lactate, in conjunction with glucose, modulates intracellular Ca²⁺ concentration in round spermatids and pachytene spermatocytes. In spite of this central role of L-lactate in spermatogenic cell physiology, the mechanism of L-lactate transport, as well as possible differentiation during spermatogenesis, has not been studied in these cells. By measuring radioactive L-lactate transport and intracellular pH (pH_i) changes with pH_i fluorescent probes, we show that these cells transport L-lactate using monocarboxylate-H⁺ transport (MCT) systems. RT-PCR, in situ mRNA hybridization, and immunocyto- and immunohistochemistry data show that pachytene spermatocytes express mainly the MCT1 and MCT4 isoforms of the transporter (intermediate- and low-affinity transporters, respectively), while round spermatids, besides MCT1 and MCT4, also show expression of the MCT2 isoform (high-affinity transporter). These molecular data are consistent with the kinetic data of L-lactate transport in these cells demonstrating at least two transport components for L-lactate. These separate transport components reflect the ability of these cells to switch between the generation of glycolytic L-lactate in the presence of external glucose and the use of L-lactate when this substrate is available in the external environment. The supply of these substrates is regulated by the hormonal control of Sertoli cell glycolytic activity. cell differentiation; seminiferous tubules; spermatogenesis; testicle; meiosis     

Transport of L-Lactate, D-Lactate, and glycolate by the LldP and GlcA membrane carriers of Escherichia coli.

To examine the substrate specificity of the membrane transport carriers LldP (L-lactate permease) and GlcA (glycolate permease) of Escherichia coli, a mutant strain lacking their structural genes and blocked in the metabolism of the tested substrates was constructed and transformed with a plasmid bearing either the lldP or the glcA gene. Each transformant acquired the ability to accumulate L-lactate, D-lactate, and glycolate against a high concentration gradient. Substrate accumulation was inhibited by carbonyl cyanide m-chlorophenylhydrazone, a hydrophobic proton conductor that dissipates proton motive force. Competition of (14)C-L-lactate transport by nonradioactive L-lactate, D-lactate, and glycolate in LldP synthesizing cells and competition of (14)C-glycolate transport by the same three substrates in GlcA synthesizing cells showed that both carriers effectively transported all three substrates with a K(i) value ranging from 10 to 20 microM. D-Lactate does not appear to have a permease of its own. Utilization of the compound depends mainly on LldP.     

Mitochondrial dysfunction and cancer metastasis

Mitochondria have an essential role in powering cells by generating ATP following the metabolism of pyruvate derived from glycolysis. They are also the major source of generating reactive oxygen species (ROS), which have regulatory roles in cell death and proliferation. Mutations in mitochondrial DNA (mtDNA) and dysregulation of mitochondrial metabolism have been frequently described in human tumors. Although the role of oxidative stress as the consequence of mtDNA mutations and/or altered mitochondrial functions has been demonstrated in carciongenesis, a causative role of mitochondria in tumor progression has only been demonstrated recently. Specifically, the subject of this mini-review focuses on the role of mitochondria in promoting cancer metastasis. Cancer relapse and the subsequent spreading of cancer cells to distal sites are leading causes of morbidity and mortality in cancer patients. Despite its clinical importance, the underlying mechanisms of metastasis remain to be elucidated. Recently, it was demonstrated that mitochondrial oxidative stress could actively promote tumor progression and increase the metastatic potential of cancer cells. The purpose of this mini-review is to summarize current investigations of the roles of mitochondria in cancer metastasis. Future development of diagnostic and therapeutic strategies for patients with advanced cancer will benefit from the new knowledge of mitochondrial metabolism in epithelial cancer cells and the tumor stroma.     

线粒体铁代谢与人类疾病的研究进展

线粒体铁代谢的研究主要包括两个方面：铁在胞质和线粒体之间的转运和调控;铁硫簇和血红素在线粒体内的合成与转运。目前认为线粒体铁的转入主要是与mitoferrinl／2（MFRNl和MFRN2）和ABCBl0有关,运出可能与ABCB6和／或ABCB7有关,转运和调控的具体机制不是很清楚,推测与某种含有铁硫簇的信号分子有关。哺乳动物铁硫簇的合成可以发生在胞质和线粒体内,但以线粒体为主;真核生物中与铁硫簇合成相关的蛋白达二十多种,其中FXN、ISCS、ISDll和ISCU及其同系物被认为是核心组分。血红素的合成起始和终止发生在线粒体内,终止步骤为亚铁螯合酶将铁插入原卟啉IX,该酶活性又依赖于铁硫簇。因此,铁硫簇的合成与调控是线粒体铁代谢的核心,也是整个细胞铁运作的核心。本文主要围绕线粒体铁代谢特别是铁硫簇的合成异常引起的疾病做一简单的综述。     

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.     

Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1

We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure D- or L-lactate. Several pta mutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to D-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in ppc made the mutant produce D-lactate like a homofermentative lactic acid bacterium. When the pta ppc double mutant was grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of D-lactate production, more than 62.2 g of D-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous L-lactate pathway, an L-lactate dehydrogenase gene from Lactobacillus casei was introduced into a pta ldhA strain which lacked phosphotransacetylase and D-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to L-lactate as the major fermentation product, and up to 45 g of L-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of D-lactate, an indigenous fermentation product, or to the production of L-lactate, a nonindigenous fermentation product.     

Selective utilization of L-isomer of lactate in the smooth muscle of the guinea pig taenia caeci

Utilization of D- and L-lactate in the isolated intestinal smooth muscle of the guinea pig taenia caeci was examined by measuring contractile tension, oxygen consumption, and adenosine triphosphate (ATP) and creatine phosphate (PCr) concentrations. In the absence of glucose in the medium, muscle contraction induced by a high concentration of K+ was inhibited and the rate of oxygen consumption and the concentrations of ATP and PCr were decreased. Addition of glucose, L-lactate, and D,L-lactate, but not D-lactate, led to recovery of muscle contraction, rate of oxygen consumption, and ATP and PCr concentrations when the tissue had been incubated in the high K+, glucose-free solution. These results suggest that the isolated guinea pig taenia caeci selectively utilizes the L-isomer of lactate as a substrate for energy metabolism.     

Competence regulation by oxygen availability and by Nox is not related to specific adjustment of central metabolism in Streptococcus pneumoniae

In Streptococcus pneumoniae oxygen availability is a major determinant for competence development in exponentially growing cultures. NADH oxidase activity is required for optimal competence in cultures grown aerobically. The implication of oxidative metabolism and more specifically of Nox on central metabolism has been examined. Glycolytic flux throughout exponential growth revealed homolactic fermentation with a lactate production/glucose utilization ratio close to 2, whatever the aerobiosis level of the culture. Loss-of-function mutations in nox, which encodes NADH oxidase, did not change this trait. Consistently, mRNA levels of glyceraldehyde-3-phosphate dehydrogenase, L-lactate dehydrogenase, pyruvate oxidase, and NADH oxidase remained comparable to wild-type levels, as did the specific activities of key enzymes which control central metabolism. Competence regulation by oxygen involving the NADH oxidase activity is not due to significant modification of carbon flux through glycolysis. Failure to obtain loss-of-function mutation in L-ldh, which encodes the L-lactate dehydrogenase, indicates its essential role in pneumococci whatever their growth status.