Mitochondrial Lactate Dehydrogenase Research Articles

Direct mitochondrial import of lactate supports resilient carbohydrate oxidation.

Lactate is the highest turnover circulating metabolite in mammals. While traditionally viewed as a waste product, lactate is an important energy source for many organs, but first must be oxidized to pyruvate for entry into the tricarboxylic acid cycle (TCA cycle). This reaction is thought to occur in the cytosol, with pyruvate subsequently transported into mitochondria via the mitochondrial pyruvate carrier (MPC). Using 13C stable isotope tracing, we demonstrated that lactate is oxidized in the myocardial tissue of mice even when the MPC is genetically deleted. This MPC-independent lactate import and mitochondrial oxidation is dependent upon the monocarboxylate transporter 1 (MCT1/Slc16a1). Mitochondria isolated from the myocardium without MCT1 exhibit a specific defect in mitochondrial lactate, but not pyruvate, metabolism. The import and subsequent mitochondrial oxidation of lactate by mitochondrial lactate dehydrogenase (LDH) acts as an electron shuttle, generating sufficient NADH to support respiration even when the TCA cycle is disrupted. In response to diverse cardiac insults, animals with hearts lacking MCT1 undergo rapid progression to heart failure with reduced ejection fraction. Thus, the mitochondrial import and oxidation of lactate enables carbohydrate entry into the TCA cycle to sustain cardiac energetics and maintain myocardial structure and function under stress conditions.

Revisiting the Warburg Effect with Focus on Lactate.

Rewired metabolism is acknowledged as one of the drivers of tumor growth. As a result, aerobic glycolysis, or the Warburg effect, is a feature of many cancers. Increased glucose uptake and glycolysis provide intermediates for anabolic reactions necessary for cancer cell proliferation while contributing sufficient energy. However, the accompanying increased lactate production, seemingly wasting glucose carbon, was originally explained only by the need to regenerate NAD+ for successive rounds of glycolysis by the lactate dehydrogenase (LDH) reaction in the cytosol. After the discovery of a mitochondrial LDH isoform, lactate oxidation entered the picture, and lactate was recognized as an important oxidative fuel. It has also been revealed that lactate serves a variety of signaling functions and helps cells adapt to the new environment. Here, we discuss recent findings on lactate metabolism and signaling in cancer while attempting to explain why the Warburg effect is adopted by cancer cells.

Lactate Utilization Provides a Metabolic Escape to Resist the Antileukemic Activity of BET Inhibition in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a molecularly heterogeneous malignant disease of the bone marrow resulting from arrest of hematopoietic precursors and uncontrolled proliferation of myeloblasts. Inhibition of the BET-family co-activator BRD4 (BETi) shows encouraging pre-clinical activity but limited clinical activity as a single agent. We previously demonstrated that MYC suppression by BETi induces metabolic and mitochondrial changes in AML blasts leading to a collapse in mitochondrial respiration. AML blasts that are resistant to BETi undergo comparable mitochondrial respiration despite impaired glycolysis and glutaminolysis. The metabolic pathway allowing BETi-resistant AML blasts to maintain metabolic integrity is unclear. Therefore, to identify how AML blast cells metabolically bypass BET inhibition, we used two different cell lines: BETi-resistant MOLM-13 and BETi-susceptible MV-4-11 cells. AML cell lines were treated with BETi for 48 hr and global untargeted metabolic mass spectrometry revealed that glycolysis, glutaminolysis, and fatty acid metabolism were not leveraged to maintain tricarboxylic acid (TCA) cycle activity (Fig. 1A). Rather, an increased NADH/NAD+ ratio and intracellular lactate accumulation suggested that lactate was being utilized as an alternative carbon source to sustain mitochondrial respiration. In support, MOLM-13 cells treated with BETi accumulated higher levels of mitochondrial lactate dehydrogenase, which preferentially converts lactate into pyruvate to fuel the TCA cycle. The heightened capacity of MOLM-13 cells to utilize lactate better maintained cellular viability but failed to support cellular replication, suggesting that lactate utilization promotes cellular senescence during BET inhibition. Therefore, we employed three chemical inhibitors of lactate utilization to couple with BETi: AZD3965 to prevent extracellular lactate uptake, UK5099 to block lactate import into the mitochondria, and oxamate to broadly impair lactate dehydrogenase activity. All three chemical inhibitors of lactate utilization were sufficient to render MOLM-13 cells susceptible to BET inhibition, as mitochondrial respiration, ATP production, and cellular viability were decreased to levels comparable to MV-4-11 cells. A similar correlation was observed in patient samples where AML blasts with increased resistance to BETi were predisposed to utilize lactate as an alternative carbon source and rendered susceptible to BET inhibition with lactate utilization inhibitors. Furthermore, these results were translated in vivo as low-dose combination of BETi and oxamate significantly reduced burdens of BETi-resistant cells in murine models (Fig. 1B). These results demonstrate that some AML blasts metabolically adapt to overcome BET inhibition by consuming lactate but that combinations of BETi and lactate utilization inhibitors can stymie this resistance. Recognizing the metabolic switching to utilize lactate in AML therapy resistance, and studying lactate utilization inhibitors in clinical settings, especially in BETi-based therapies should be considered. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Role of Mitochondrial Membrane Potential and Lactate Dehydrogenase A in Apoptosis.

Apoptosis is a programmed cell death that occurs due to the production of several catabolic enzymes. During this process, several morphological and biochemical changes occur in mitochondria, the main organelle in the cell that participates in apoptosis and controls apoptotic pathways. During apoptosis, cytochrome c is released from mitochondria, and different proteins activate caspase cascades that carry out the cell towards the death process. Apoptosis mainly occurs due to p53 protein that allows the abnormal cells to proliferate. Bcl-2 and Bcl-xl are two anti-apoptotic members of the protein family that prevents apoptosis. The membrane potential of mitochondria decreases by the opening of the permeability transition pore (PTP). These PTP are formed by the binding of Bax with adenine nucleotide translocator (ANT) and cause depolarization in the membrane. The depolarization releases apoptogenic factors (cytochrome c) that result in the loss of oxidative phosphorylation. Knockdown in lactate dehydrogenase (LDH) is the cause of the decrease in mitochondrial membrane potential elevating the levels of reactive oxygen species (ROS) and Bax. Consequently, causing an increase in the release of cytochrome c that ultimately leads to apoptosis. In this review, we have summarized the combined effect of mitochondrial membrane potential and LDH enzyme that triggers apoptosis in cells and their role in the mechanism of apoptosis.

An Upstream Open Reading Frame in Phosphatase and Tensin Homolog Encodes a Circuit Breaker of Lactate Metabolism

The metabolic role of micropeptides generated from untranslated regions remains unclear. Here we describe MP31, a micropeptide encoded by the upstream open reading frame (uORF) of phosphatase and tensin homolog (PTEN) acting as a "circuit breaker" that limits lactate-pyruvate conversion in mitochondria by competing with mitochondrial lactate dehydrogenase (mLDH) for nicotinamide adenine dinucleotide (NAD+). Knocking out the MP31 homolog in mice enhanced global lactate metabolism, manifesting as accelerated oxidative phosphorylation (OXPHOS) and increased lactate consumption and production. Conditional knockout (cKO) of MP31 homolog in mouse astrocytes initiated gliomagenesis and shortened the overall survival of the animals, establishing a tumor-suppressing role for MP31. Recombinant MP31 administered intraperitoneally penetrated the blood-brain barrier and inhibited mice GBM xenografts without neurological toxicity, suggesting the clinical implication and application of this micropeptide. Our findings reveal a novel mode of MP31-orchestrated lactate metabolism reprogramming in glioblastoma.

Phytochemical Screening and Effect of Cymodocea serrulata on HepG2-Human Hepatocellular Carcinoma Cell Line

Cymodocea serrulata, a seagrass commonly known as karumbupassi has been used as a food and also as a medicine by coastal region people and by fishermen while traveling in the sea. It is used as a tranquilizer for babies as it has a soothing quality, it helps during pregnancy and against cough and malaria. C.serrulata is seen abundant in South Indian coastal region. Although there is a report on the antibacterial, antioxidant, and anti-inflammatory property of C.serrulata, there are no evident details on phyto-compounds present in it. Invitro antiproliferative test was performed by MTT (methylthiozoltetrazolium) assay method against HePG2 celline of liver cancer cells. MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on reduction of the yellow colored water soluble tetrazolium dye MTT to formazan crystals. Mitochondrial lactate dehydrogenase produced by liver cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple color, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570nm.

Malate-aspartate shuttle promotes l-lactate oxidation in mitochondria.

Metabolism in cancer cells is rewired to generate sufficient energy equivalents and anabolic precursors to support high proliferative activity. Within the context of these competing drives aerobic glycolysis is inefficient for the cancer cellular energy economy. Therefore, many cancer types, including colon cancer, reprogram mitochondria-dependent processes to fulfill their elevated energy demands. Elevated glycolysis underlying the Warburg effect is an established signature of cancer metabolism. However, there are a growing number of studies that show that mitochondria remain highly oxidative under glycolytic conditions. We hypothesized that activities of glycolysis and oxidative phosphorylation are coordinated to maintain redox compartmentalization. We investigated the role of mitochondria-associated malate-aspartate and lactate shuttles in colon cancer cells as potential regulators that couple aerobic glycolysis and oxidative phosphorylation. We demonstrated that the malate-aspartate shuttle exerts control over NAD+ /NADH homeostasis to maintain activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation of glycolytic l-lactate in mitochondria. The elevated glycolysis in cancer cells is proposed to be one of the mechanisms acquired to accelerate oxidative phosphorylation.

183 - Mitochondria-localized lactate dehydrogenase is not a biologically significant contributor to bioenergetic function in striated muscle

Previous studies indicate that mitochondrial lactate dehydrogenase (mLDH) might be a significant contributor to metabolism. The presence of mLDH could provide mitochondria with a higher capacity to generate reducing equivalents for respiration, especially during exercise when circulating lactate levels are high. Furthermore, mitochondrial lactate oxidation could support cardiac output or skeletal muscle function, both of which contribute to exercise capacity. We measured lactate-, pyruvate-, and glutamate-supported respiration in mitochondria isolated from heart and skeletal muscle of sedentary, acutely exercised, and exercise-adapted mice. We modeled the acute effects of exercise by subjecting male FVB/NJ mice to one, 60 min bout of treadmill running. Exercise adaptation was modeled with progressive treadmill training at 40-60 min/d for 2 weeks, with adaptation determined by measuring exercise capacity and indices of cardiac growth. We performed immunoblotting to assess relative abundance of LDH isoforms in isolated mitochondria. Two weeks of treadmill running increased running distance by 1.3-fold. Compared with sedentary mice, exercise training increased cardiac mass by 15% (n=5/group, p<0.01). Cardiac mitochondria energized with 5 mM pyruvate+2.5 mM malate, and skeletal muscle mitochondria provided with 5 mM glutamate+2.5 mM malate, showed approximately 10-fold higher respiration rates compared with mitochondria provided 5 mM lactate+2.5 mM malate. Exercise training did not significantly affect respiration on either substrate. We performed similar studies on mitochondria isolated immediately after one intense bout of exercise. In both sedentary and exercised conditions, cardiac and skeletal muscle mitochondria provided with lactate showed 10-fold lower respiration rates than those supplied with pyruvate or glutamate (n=3/group). Western blotting indicated low levels of LDHB in mitochondrial fractions from heart and low levels of LDHA in mitochondrial fractions from skeletal muscle. Lactate metabolism in striated muscle is primarily a cytosolic phenomenon; mLDH is of minimal biological significance.

The cytotoxic effects of propolis on breast cancer cells involve PI3K/Akt and ERK1/2 pathways, mitochondrial membrane potential, and reactive oxygen species generation

Propolis has been extensively used to improve health and prevent inflammatory diseases. Different types of Cuban propolis (red, brown and yellow) have been documented. The purpose of this research was to investigate the cytotoxic effects of Cuban red propolis (CP) on MDA MB-231 cell line, since breast cancer is considered one of the most common causes of mortality among women. Antiproliferative and cytotoxic activity of CP against MDA MB-231 cells were determined by the 3-[4,5-dimethylth-iazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assays. Apoptosis/necrosis, involvement of PI3K/Akt and ERK1/2 pathways, mitochondrial membrane potential and expression of genes were investigated. CP extract exhibited antiproliferative and cytotoxic effects on MDA MB-231 cells, what may be probably related to PI3K/Akt and ERK1/2 pathways. A decreased expression of apoptosis-related genes (TP53, CASP3, BAX and P21) was seen, whereas the expressions of BCL-2, BCL-XL, NOXA and PUMA were unaffected. CP extract induced mitochondrial dysfunction and LDH release, what indicated cell necrosis associated with reactive oxygen species production and decreased cell migration. Our findings provide a basis for future investigation of chemopreventive and/or therapeutic studies against apoptosis-resistant breast cancer, in animals and humans.

Osteoblasts MC3T3-E1 Response in 2D and 3D Cell Cultures Models to High Carbon Content CoCr Alloy Particles. Effect of Metallic Particles on Vimentin Expression

The study of the biocompability of the metallic materials is a priority treating to avoid the osteolysis and aseptic loosening of prosthesis. Wear debris is considered one of the main factors responsible for aseptic loosening of orthopedic endoprostheses. We examined the response of mouse osteoblasts MC3T3-E1 to high carbon cobalt-chrome (HCCoCr) particles obtained a) from wear-corrosion assays on a pin-on-disk tribometer using as pair an alumina ball and a disc of a HCCoCr alloy, and b) HCCoCr bulk particles obtained by nitrogen gas atomization from an alloy used in clinic for prostheses application. Mitochondrial activity and lactate dehydrogenase activity assayed in 2D and 3D osteoblasts cell culture models were used to evaluate the cellular response to size, shape, and chemical composition of the metallic particles. 2D cell model was used to study the direct interaction of cells with particles and 3D cell cultures was used to more closely mimic in vivo conditions. The results showed that vimentin was overexpressed in the 2D osteoblasts cultures in presence of metal particles. This might be related to the appearance of pseudotumor in the peri-prosthetic vicinity described in some implanted patients.

Activities of cytochrome c oxidase and mitochondrial lactate dehydrogenase isozymes and Cox1, Cox2, Cox4, and Cox6 gene subunit expression in cold adaptation of Salmo trutta L.

Characteristic changes in some parameters of white muscle mitochondria (mitochondrial volume, activity of cytochrome c oxidase (COX, EC 1.9.3.1) and the level of Cox1, Cox2, Cox4, and Cox6 subunit gene expression and activity and kinetic characteristics of mitochondrial lactate dehydrogenase isozymes (mtLDH, EC 1.1.1.27)) in adaptation to seasonal decrease in temperature from 16 to 6°C of one-year juvenile brown trout Salmo trutta L. from rivers of Lake Onega basin were investigated. A 1.5-fold increase in the activity of COX, and the increase in the levels of gene expression of both Cox4 and Cox6 nuclear subunits and increased activity of mitochondrial LDH isozymes, which have a low affinity towards lactate, has been shown. The possible role of nuclear and mitochondrial subunits of cytochrome c oxidase in improving the efficiency of the enzyme in molecule biogenesis and further modulation of its activity, as well as regulation of pyruvate formation to maintain the required rate of oxidative phosphorylation in mitochondria under low temperature were discussed.

Synthesis, Characterization and Molecular Docking of Novel Bioactive Thiazolyl-Thiazole Derivatives as Promising Cytotoxic Antitumor Drug.

Reactions of ethylidenethiocarbohydrazide with hydrazonoyl halides gave 1,3-thiazole or 1,3,4-thiadiazole derivatives according to the type of hydrazonoyl halides. Treatment of ethylidenethiosemicarbazide with hydrazonoyl halides and dimethylacetylene dicarboxylate (DMAD) afforded the corresponding arylazothiazoles and 1,3-thiazolidin-4-one derivatives, respectively. The structures of the synthesized products were confirmed by IR, 1H-NMR, 13C-NMR and mass spectral techniques. The cytotoxic activity of the selected products against the Hepatic carcinoma cell line (Hepg-2) was determined by MTT assay indicating a concentration dependent cellular growth inhibitory effect, especially for compounds 14c and 14e. The dose response curves indicated the IC50 (the concentration of test compounds required to kill 50% of cell population) were 0.54 μM and 0.50 μM, respectively. Confocal laser scanning imaging of the treated cells stained by Rhodamin 123 and Acridine orange dyes confirmed that the selected compounds inhibit the mitochondrial lactate dehydrogenase enzymes. The binding mode of the active compounds was interpreted by a molecular docking study. The obtained results revealed promising cytotoxic activity.

Cerebral glycolysis: a century of persistent misunderstanding and misconception.

Since its discovery in 1780, lactate (lactic acid) has been blamed for almost any illness outcome in which its levels are elevated. Beginning in the mid-1980s, studies on both muscle and brain tissues, have suggested that lactate plays a role in bioenergetics. However, great skepticism and, at times, outright antagonism has been exhibited by many to any perceived role for this monocarboxylate in energy metabolism. The present review attempts to trace the negative attitudes about lactate to the first four or five decades of research on carbohydrate metabolism and its dogma according to which lactate is a useless anaerobic end-product of glycolysis. The main thrust here is the review of dozens of scientific publications, many by the leading scientists of their times, through the first half of the twentieth century. Consequently, it is concluded that there exists a barrier, described by Howard Margolis as “habit of mind,” that many scientists find impossible to cross. The term suggests “entrenched responses that ordinarily occur without conscious attention and that, even if noticed, are hard to change.” Habit of mind has undoubtedly played a major role in the above mentioned negative attitudes toward lactate. As early as the 1920s, scientists investigating brain carbohydrate metabolism had discovered that lactate can be oxidized by brain tissue preparations, yet their own habit of mind redirected them to believe that such an oxidation is simply a disposal mechanism of this “poisonous” compound. The last section of the review invites the reader to consider a postulated alternative glycolytic pathway in cerebral and, possibly, in most other tissues, where no distinction is being made between aerobic and anaerobic glycolysis; lactate is always the glycolytic end product. Aerobically, lactate is readily shuttled and transported into the mitochondrion, where it is converted to pyruvate via a mitochondrial lactate dehydrogenase (mLDH) and then is entered the tricarboxylic acid (TCA) cycle.

Synthesis, characterization, and pharmacological evaluation of some novel thiadiazoles and thiazoles incorporating pyrazole moiety as anticancer agents

Two series of novel 2-[[1-(5-methyl-1-phenyl-5-substituted-1H-pyrazol-4-yl)ethylidene]hydrazono]-3-phenyl-2,3-dihydro-1,3,4-thiadiazole derivatives and 2-[5-(4-chlorophenyl)-5′-methyl-1′-phenyl-3,4-dihydro-1′H,2H-[3,4′-bipyrazol]-2-yl]-4-substituted-5-(phenyldiazenyl)thiazole derivatives were prepared from reaction of hydrazonoyl halides with methyl 2-[1-(5-methyl-1-phenyl-1H-pyrazol-4-yl)ethylidene]hydrazine-carbodithioate and thiosemicarbazide derivative, respectively. The newly synthesized derivatives were elucidated by elemental analysis, spectral data, and alternative synthetic routes, whenever possible. The anti-cancer activity of the selected products against the breast carcinoma cell line MCF-7 was determined by WST-1 assay indicating concentration-dependent cellular growth inhibitory effect especially for three compounds with dose response curves indicating IC 50 values of 21.3 ± 0.72, 21.3 ± 0.72, and 23.56 ± 0.81 µg cm−3, respectively. Confocal laser scanning imaging of the treated cells stained by rhodamin 123 and acridine orange dyes confirms that the selected compounds inhibit the mitochondrial lactate dehydrogenase enzymes. The obtained results revealed promising anticancer activity.

Mitochondrial lactate metabolism is involved in antioxidative defense in human astrocytoma cells

Although lactate has traditionally been known to be an end product of anaerobic metabolism, recent studies have revealed its disparate biological functions. Oxidative energy production and cell signaling are two important roles assigned to this monocarboxylic acid. Here we demonstrate that mitochondrial lactate metabolism to pyruvate mediated by lactate dehydrogenase (LDH) in a human astrocytic cell line is involved in antioxidative defense. The pooling of this α-ketoacid helps to detoxify reactive oxygen species, with the concomitant formation of acetate. In-gel activity assays following blue native PAGE electrophoresis were utilized to demonstrate the increase in mitochondrial LDH activity coupled to the decrease in pyruvate dehydrogenase activity in the cells challenged by oxidative stress. The enhanced production of pyruvate with the concomitant formation of acetate in astrocytoma cells was monitored by high-performance liquid chromatography. The ability of pyruvate to fend off oxidative stress was visualized by fluorescence microscopy with the aid of the dye 2',7'-dichlorodihydrofluorescein diacetate. Immunoblotting helped confirm the presence of elevated levels of LDH in cells exposed to oxidative stress, and recovery experiments were performed with pyruvate to diminish the oxidative burden on the astrocytoma. The acetate, generated as a consequence of the antioxidative attribute of pyruvate, was subsequently channeled toward the production of lipids, a process facilitated by the upregulation in activity of acetyl-CoA synthetase and acetyl-CoA carboxylase, as demonstrated by in-gel activity assays. The mitochondrial lactate metabolism mediated by LDH appears to play an important role in antioxidative defence in this astrocytic system.

Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO2) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO2. However, subsequent addition of NAD(+) significantly increased JO2, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD(+)-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.

The key involvement of estrogen receptor β and G-protein-coupled receptor 30 in the neuroprotective action of daidzein

Phytoestrogens have received considerable attention because they provide an array of beneficial effects, such as neuroprotection. To better understand the molecular and functional link between phytoestrogens and classical as well as membrane estrogen receptors (ERs), we investigated the effect of daidzein on the glutamate-mediated apoptotic pathway. Our study demonstrated that daidzein (0.1–10μM) inhibited the pro-apoptotic and neurotoxic effects caused by glutamate treatment. Hippocampal, neocortical and cerebellar tissues responded to the inhibitory action of daidzein on glutamate-activated caspase-3 and lactate dehydrogenase (LDH) release in a similar manner. Biochemical data were supported at the cellular level by Hoechst 33342 and calcein AM staining. The sensitivity of neuronal cells to daidzein-mediated protection was most prominent in hippocampal cultures at an early stage of development 7th day in vitro. A selective estrogen receptor β (ERβ) antagonist, 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5,-a]pyrimidin-3-yl]phenol (PHTPP), and a selective G-protein-coupled receptor 30 (GPR30) antagonist, 3aS∗,4R∗,9bR∗)-4-(6-Bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G15), reversed the daidzein-mediated inhibition of glutamate-induced loss of membrane mitochondrial potential, caspase-3 activity, and LDH release. A selective ERα antagonist, methyl-piperidino-pyrazole (MPP), did not influence any anti-apoptotic effect of daidzein. However, a high-affinity estrogen receptor antagonist, 7α,17β-[9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol (ICI) 182,780, and a selective GPR30 agonist, (±)-1-[(3aR∗,4S∗,9bS∗)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-8-yl]-ethanone (G1), intensified the protective action of daidzein against glutamate-induced loss of membrane mitochondrial potential and LDH release. In siRNA ERβ- and siRNA GPR30-transfected cells, daidzein did not inhibit the glutamate-induced effects. Twenty-four hour exposure to glutamate did not affect the cellular distribution of ERβ and GPR30, but caused greater than 100% increase in the levels of the receptors. Co-treatment with daidzein decreased the level of ERβ without significant changing of the GPR30 protein level.Here, we elucidated neuroprotective effects of daidzein at low micromolar concentrations and demonstrated that the phytoestrogens may exert their effects through novel extranuclear GPR30 and the classical transcriptionally acting ERβ. These studies uncover key roles of the ERβ and GPR30 intracellular signaling pathways in mediating the anti-apoptotic action of daidzein and may provide insight into new strategies to treat or prevent neural degeneration.

Rat extraocular muscles use lactate as mitochondrial substrate

Extraocular muscles (EOMs) have unique adaptations to match the demands imposed by constant activity. We previously showed that lactate is a potential energy source in EOMs, not metabolic waste. Here, we tested the hypothesis that EOM mitochondria can use lactate. We isolated mitochondria from EOMs of adult male Sprague Dawley rats to measure respiration (O2 consumption) by polarography. State 2 respiration started with glutamate/malate, lactate/malate or lactate/malate plus NAD+. ADP initiated state 3 respiration. Oligomycin, an ATP synthase inhibitor, was used to estimate state 4. Uncoupled respiration, state 5, was determined by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. A respiratory control ratio (state 3/state 4) ≥ 4 was evidence of viable mitochondria. We determined lactate dehydrogenase (LDH) content by western blot. There was no significant difference in O2 consumption with lactate/malate or lactate/malate plus NAD+ when compared to glutamate/malate for states 2, 4, and 5 (p≥ 0.05). However, state 3 was higher with glutamate/malate compared to lactate/malate (p=0.004). There were no differences in state 3 with other substrates. LDH is present in EOM mitochondria. We conclude that rat EOMs can use lactate as a metabolic substrate for mitochondrial respiration and that mitochondrial LDH is likely responsible for oxidation of lactate to pyruvate. Supported by R01 EY012998 to FHA.

Aerobic production and utilization of lactate satisfy increased energy demands upon neuronal activation in hippocampal slices and provide neuroprotection against oxidative stress

Ever since it was shown for the first time that lactate can support neuronal function in vitro as a sole oxidative energy substrate, investigators in the field of neuroenergetics have been debating the role, if any, of this glycolytic product in cerebral energy metabolism. Our experiments employed the rat hippocampal slice preparation with electrophysiological and biochemical methodologies. The data generated by these experiments (a) support the hypothesis that lactate, not pyruvate, is the end-product of cerebral aerobic glycolysis; (b) indicate that lactate plays a major and crucial role in affording neural tissue to respond adequately to glutamate excitation and to recover unscathed post-excitation; (c) suggest that neural tissue activation is accompanied by aerobic lactate and NADH production, the latter being produced when the former is converted to pyruvate by mitochondrial lactate dehydrogenase (mLDH); (d) imply that NADH can be utilized as an endogenous scavenger of reactive oxygen species (ROS) to provide neuroprotection against ROS-induced neuronal damage.

Mitochondrial Lactate Dehydrogenase Is Involved in Oxidative-Energy Metabolism in Human Astrocytoma Cells (CCF-STTG1)

Lactate has long been regarded as an end product of anaerobic energy production and its fate in cerebral metabolism has not been precisely delineated. In this report, we demonstrate, for the first time, the ability of a human astrocytic cell line (CCF-STTG1) to consume lactate and to generate ATP via oxidative phosphorylation. 13C-NMR and HPLC analyses aided in the identification of tricarboxylic acid (TCA) cyle metabolites and ATP in the astrocytic mitochondria incubated with lactate. Oxamate, an inhibitor of lactate dehydrogenase (LDH), abolished mitochondrial lactate consumption. Electrophoretic and fluorescence microscopic analyses helped localize LDH in the mitochondria. Taken together, this study implicates lactate as an important contributor to ATP metabolism in the brain, a finding that may significantly change our notion of how this important organ manipulates its energy budget.