Lactate Dehydrogenase Protein Levels Research Articles

Exposure to toxicologically relevant atrazine concentrations impair the glycolytic function of mouse Sertoli cells through the downregulation of lactate dehydrogenase

Atrazine (ATZ), a widely used herbicide with potent endocrine-disrupting properties, has been implicated in hormonal disturbances and fertility issues. Sertoli cells (SCs) play a crucial role in providing mechanical and nutritional support of spermatogenesis. Herein, we aimed to study the effects of environmentally relevant ATZ concentrations on the nutritional support of spermatogenesis provided by SCs. For that, mouse SCs (TM4) were exposed to increasing ATZ concentrations (in μg/L: 0.3, 3, 30, 300, or 3000). After 24 h, cellular proliferation and metabolic activity were assessed. Mitochondrial activity and endogenous reactive oxygen species (ROS) production were evaluated using JC-1 and CM-H2DCFDA probes, respectively. We also analyzed protein levels of lactate dehydrogenase (LDH) using Western Blot and live cells glycolytic function through Seahorse XF Glycolysis Stress Test Kit. ATZ exposure decreased the activity of oxidoreductases in SCs, suggesting a decreased metabolic activity. Although ATZ is reported to induce oxidative stress, we did not observe alterations in mitochondrial membrane potential and ROS production across all tested concentrations. When we evaluated the glycolytic function of SCs, we observed that ATZ significantly impaired glycolysis and the glycolytic capacity at all tested concentrations. These results were supported by the decreased expression of LDH in SCs. Overall, our findings suggest that ATZ impairs the glycolytic function of SCs through LDH downregulation. Since lactate is the preferential energetic substrate for germ cells, exposure to ATZ may detrimentally impact the nutritional support crucial for spermatogenesis, hinting for a relationship between ATZ exposure and male infertility.

Evidence for Hypoxia-Induced Shift in ATP Production from Glycolysis to Mitochondrial Respiration in Pulmonary Artery Smooth Muscle Cells in Pulmonary Arterial Hypertension.

The metabolic state of pulmonary artery smooth muscle cells (PASMCs) from patients with pulmonary arterial hypertension (PAH) is not well understood. In this study, we examined the balance between glycolysis and mitochondrial respiration in non-PAH-PASMCs and PAH-PASMCs under normoxia and hypoxia. We investigated the enzymes involved in glycolysis and mitochondrial respiration, and studied the two major energy-yielding pathways (glycolysis and mitochondrial respiration) by measuring extracellular acidification rate (ECAR) and cellular oxygen consumption rate (OCR) using the Seahorse extracellular flux technology. Under both normoxia and hypoxia, the mRNA and protein levels of pyruvate dehydrogenase kinase 1 and pyruvate dehydrogenase were increased in PAH-PASMCs compared with non-PAH-PASMCs. The mRNA and protein levels of lactate dehydrogenase, as well as the intracellular lactate concentration, were also increased in PAH-PASMCs compared with non-PAH-PASMCs under normoxia. However, these were not significantly increased in PAH-PASMCs compared with non-PAH-PASMCs under hypoxia. Under normoxia, ATP production was significantly lower in PAH-PASMCs (59 ± 5 pmol/min) than in non-PAH-PASMCs (70 ± 10 pmol/min). On the other hand, ATP production was significantly higher in PAH-PASMCs (31 ± 5 pmol/min) than in non-PAH-PASMCs (14 ± 3 pmol/min) under hypoxia. There is an underlying change in the metabolic strategy to generate ATP production under the challenge of hypoxia.

The Warburg effect as a therapeutic target for bladder cancers and intratumoral heterogeneity in associated molecular targets.

Bladder cancer is the 10th most common cancer worldwide. For muscle‐invasive bladder cancer (MIBC), treatment includes radical cystectomy, radiotherapy, and chemotherapy; however, the outcome is generally poor. For non–muscle‐invasive bladder cancer (NMIBC), tumor recurrence is common. There is an urgent need for more effective and less harmful therapeutic approaches. Here, bladder cancer cell metabolic reprogramming to rely on aerobic glycolysis (the Warburg effect) and expression of associated molecular therapeutic targets by bladder cancer cells of different stages and grades, and in freshly resected clinical tissue, is investigated. Importantly, analyses indicate that the Warburg effect is a feature of both NMIBCs and MIBCs. In two in vitro inducible epithelial‐mesenchymal transition (EMT) bladder cancer models, EMT stimulation correlated with increased lactate production, the end product of aerobic glycolysis. Protein levels of lactate dehydrogenase A (LDH‐A), which promotes pyruvate enzymatic reduction to lactate, were higher in most bladder cancer cell lines (compared with LDH‐B, which catalyzes the reverse reaction), but the levels did not closely correlate with aerobic glycolysis rates. Although LDH‐A is expressed in normal urothelial cells, LDH‐A knockdown by RNAi selectively induced urothelial cancer cell apoptotic death, whereas normal cells were unaffected—identifying LDH‐A as a cancer‐selective therapeutic target for bladder cancers. LDH‐A and other potential therapeutic targets (MCT4 and GLUT1) were expressed in patient clinical specimens; however, positive staining varied in different areas of sections and with distance from a blood vessel. This intratumoral heterogeneity has important therapeutic implications and indicates the possibility of tumor cell metabolic coupling.

FSMP-02. CHANGES IN GLUTAMINE METABOLISM INDUCED BY OXALOACETATE IN GLIOBLASTOMA

Anhydrous Enol-Oxaloacetate (AEO) has been shown to significantly increase survival and decrease tumor growth rates in animal models of glioblastoma and hepatocellular carcinoma. In the body, AEO is metabolized to “oxaloacetate” (OAA). Earlier, we demonstrated that AEO drastically reduced Warburg glycolysis in glioblastoma cells which was determined by the increase in pyruvate to lactate ratio and a 48.8% decrease in lactate production in 13C-labeled glucose metabolism studies. We have expanded this previous work to examine 13C-labeled glutamine metabolism. Cultured solid tumor cancer cells strongly rely on both glucose and glutamine to synthesize carbon intermediates for anaplerotic reactions. With treatment of OAA, we hypothesize that glutamine-derived OAA may be reduced which can be tracked through the use GC-MS based 13C-labeled glutamine isotopomer experiments. Patient-derived glioblastoma cells were grown in 15 mM glucose and 2 mM glutamine containing DMEM medium supplemented with 2 mM OAA for 10 days. 24 hours prior to harvesting the cells, 4 mM of [U-13C]glutamine was introduced to the medium. OAA treated cells showed significant decrease in the protein levels of lactate dehydrogenase A and C which indicates the switching off of glycolysis to support the utilization of elevated OAA levels during glutamine metabolism in the TCA cycle. 13C mass distribution analysis showed significant decrease in malate, aspartate and citrate pools (malate: 8.8%, p = 0.0098; aspartate: 9.2%, p = 0.0064; citrate: 9.5%, p = 0.0036) in their M+4 isotopomer labeling in OAA treated group compared to the control group. Decrease in lactate generation may reduce cancer proliferation, migration and invasion. Together, these data provide alternative way to modulate energy metabolism in glioblastoma using AEO treatment. Similarly, AEO treatment in other solid tumors (e.g. pancreatic ductal adenocarcinoma) may produce altered glutamine metabolism which may be of therapeutic value.

Hypoxic naked mole-rat brains use microRNA to coordinate hypometabolic fuels and neuroprotective defenses.

Naked mole-rats are among the mammalian champions of hypoxia tolerance. They evolved adaptations centered around reducing metabolic rate to overcome the challenges experienced in their underground burrows. In this study, we used next-generation sequencing to investigate one of the factors likely supporting hypoxia tolerance in naked mole-rat brains, posttranscriptional microRNAs (miRNAs). Of the 212 conserved miRNAs identified using small RNA sequencing, 18 displayed significant differential expression during hypoxia. Bioinformatic enrichment revealed that hypoxia-mediated miRNAs were suppressing energy expensive processes including de novo protein translation and cellular proliferation. This suppression occurred alongside the activation of neuroprotective and neuroinflammatory pathways, and the induction of central signal transduction pathways including HIF-1α and NFκB via miR-335, miR-101, and miR-155. MiRNAs also coordinated anaerobic glycolytic fuel sources, where hypoxia-upregulated miR-365 likely suppressed protein levels of ketohexokinase, the enzyme responsible for catalyzing the first committed step of fructose catabolism. This was further supported by a hypoxia-mediated reduction in glucose transporter 5 proteins that import fructose into the cell. Yet, messenger RNA and protein levels of lactate dehydrogenase, which converts pyruvate to lactate in the absence of oxygen, were elevated during hypoxia. Together, this demonstrated the induction of anaerobic glycolysis despite a lack of reliance on fructose as the primary fuel source, suggesting that hypoxic brains are metabolically different than anoxic naked mole-rat brains that were previously found to shift to fructose-based glycolysis. Our findings contribute to the growing body of oxygen-responsive miRNAs "OxymiRs" that facilitate natural miRNA-mediated mechanisms for successful hypoxic exposures.

Lysine-222 succinylation reduces lysosomal degradation of lactate dehydrogenase a and is increased in gastric cancer

BackgroundLysine succinylation is an emerging posttranslational modification that has garnered increased attention recently, but its role in gastric cancer (GC) remains underexplored.MethodsProteomic quantification of lysine succinylation was performed in human GC tissues and adjacent normal tissues by mass spectrometry. The mRNA and protein levels of lactate dehydrogenase A (LDHA) in GC and adjacent normal tissues were analyzed by qRT-PCR and western blot, respectively. The expression of K222-succinylated LDHA was measured in GC tissue microarray by the K222 succinylation-specific antibody. The interaction between LDHA and sequestosome 1 (SQSTM1) was measured by co-immunoprecipitation (co-IP) and proximity ligation assay (PLA). The binding of carnitine palmitoyltransferase 1A (CPT1A) to LDHA was determined by co-IP. The effect of K222-succinylated LDHA on tumor growth and metastasis was evaluated by in vitro and in vivo experiments.ResultsAltogether, 503 lysine succinylation sites in 303 proteins were identified. Lactate dehydrogenase A (LDHA), the key enzyme in Warburg effect, was found highly succinylated at K222 in GC. Intriguingly, this modification did not affect LDHA ubiquitination, but reduced the binding of ubiquitinated LDHA to SQSTM1, thereby decreasing its lysosomal degradation. We demonstrated that CPT1A functions as a lysine succinyltransferase that interacts with and succinylates LDHA. Moreover, high K222-succinylation of LDHA was associated with poor prognosis in patients with GC. Finally, overexpression of a succinylation-mimic mutant of LDHA promoted cell proliferation, invasion, and migration.ConclusionsOur data revealed a novel lysosomal pathway of LDHA degradation, which is mediated by the binding of K63-ubiquitinated LDHA to SQSTM1. Strikingly, CPT1A succinylates LDHA on K222, which thereby reduces the binding and inhibits the degradation of LDHA, as well as promotes GC invasion and proliferation. This study thus uncovers a new role of lysine succinylation and the mechanism underlying LDHA upregulation in GC.

Combined use of Diane-35 and metformin improves the ovulation in the PCOS rat model possibly via regulating glycolysis pathway

BackgroundPolycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disease with unknown pathogenesis. However, the treatment of Diane-35 combined with metformin can improve the endocrine and ovulation of PCOS. In this study, we investigated the effects of Diane-35 combined with metformin (DM) treatment on ovulation and glucose metabolism in a PCOS rat model.MethodsSprague Dawley rats were divided into 3 groups, control group, model group (PCOS group) and Diane-35 combined with metformin (PCOS + DM group). The mRNA expression levels were determined by qRT-PCR. The hormone levels were determined by enzyme-linked immunosorbent assay. Immunostaining detected the protein levels of lactate dehydrogenase A (LDH-A), pyruvate kinase isozyme M2 (PKM2) and sirtuin 1 (SIRT1) in the ovarian tissues. TNUEL assay was performed to determine cell apoptosis in the PCOS rats. The metabolites in the ovarian tissues were analyzed by liquid chromatography with tandem mass spectrometry.ResultsPCOS rats showed an increased in body weight, levels of luteinizing hormone and testosterone and insulin resistance, which was significantly attenuated by the DM treatment. The DM treatment improved disrupted estrous cycle and increased the granulosa cells of the ovary in the PCOS rats. The decreased proliferation and increased cell apoptosis of granulosa cells in the ovarian tissues of PCOS rats were significantly reversed by the DM treatment. The analysis of metabolics revealed that ATP and lactate levels were significantly decreased in PCOS rats, which was recovered by the DM treatment. Furthermore, the expression of LDH-A, PKM2 and SIRT1 was significantly down-regulated in ovarian tissues of the PCOS rats; while the DM treatment significantly increased the expression of LDH-A, PKM2 and SIRT1 in the ovarian tissues of the PCOS rats.ConclusionIn conclusion, our study demonstrated that Diane-35 plus metformin treatment improved the pathological changes in the PCOS rats. Further studies suggest that Diane-35 plus metformin can improve the energy metabolism of the ovary via regulating the glycolysis pathway. The mechanistic studies indicated that the therapeutic effects of Diane-35 plus metformin treatment in the PCOS rats may be associated with the regulation of glycolysis-related mediators including PKM2, LDH-A and SIRT1.

Xanthophyll β-Cryptoxanthin Inhibits Highly Refined Carbohydrate Diet-Promoted Hepatocellular Carcinoma Progression in Mice.

β-Cryptoxanthin (BCX) can be cleaved by both β-carotene 15,15'-oxygenase (BCO1) and β-carotene 9',10'-oxygenase (BCO2), generating biological active vitamin A and apocarotenoids. We examined whether BCX feeding could inhibit diethylnitrosamine (DEN)-initiated, highly refined carbohydrate diet (HRCD)-promoted hepatocellular carcinoma (HCC) development, dependent or independent of BCO1/BCO2 activity. Two-week-old male wild-type (WT) and BCO1-/- /BCO2-/- double knockout (DKO) mice are given a single intraperitoneal injection of DEN (25mg kg-1 body weight) to initiate hepatic carcinogenesis. At 6 weeks of age, all animals are fed HRCD (66.5% of energy from carbohydrate) with or without BCX for 24 weeks. BCX feeding increases hepatic vitamin A levels in WT mice, but not in DKO mice that shows a significant accumulation of hepatic BCX. Compared to their respective HRCD littermates, both WT and DKO fed BCX have significantly lower HCC multiplicity, average tumor size, and total tumor volume, and the steatosis scores. The chemopreventive effects of BCX are associated with increased p53 protein acetylation and decreased protein levels of lactate dehydrogenase and hypoxia-inducible factor-1α in tumors. This study suggests that BCX feeding may alleviate HRCD-promoted HCC progression by modulating the acetylation of p53, hypoxic tumor microenvironment, and glucose metabolism, independent of BCO1/BCO2.

Abstract 1127: Small molecule SIRT3 inhibitor 4′-bromo-resveratrol inhibits proliferation, promotes apoptosis and causes metabolic reprograming of human melanoma cells

Abstract Sirtuin-3 (SIRT3) is an important mitochondria NAD+-dependent deacetylase that is known to target mitochondrial proteins for deacetylation and regulates a variety of cellular functions. We have previously shown that SIRT3 was overexpressed in human melanoma cells and tissues and its genetic knockdown resulted in a significant antiproliferative response in human melanoma cells (In: AACR Annual Meeting: Proceedings; 2014 April 5-April 9, San Diego; CA, Abstract # 3516). Our data suggested that small molecule inhibitors of SIRT3 may be developed for the management of melanoma. In this study, we determined the anti-proliferative efficacy of a newly identified small molecule SIRT3 inhibitor, 4′-bromo-resveratrol, in human melanoma cell lines (G361, SK-MEL-28 and SK-MEL-2). Treatment of melanoma cells with 4-bromoresveratrol (0, 12.5, 25, 50, 100, and 200 × 10-3 mM; for 24, 48 and 72 hrs) resulted in a significant decrease in cell proliferation in a dose- and time- dependent manner. In addition, 4′-bromo-resveratrol treatment also resulted in a marked decrease in the growth, viability, clonogenic survival and migration of melanoma cells. Further, the anti-proliferative effects of 4′-bromo-resveratrol were accompanied by a marked downregulation of proliferating cell nuclear antigen (PCNA) protein as well as SIRT3 mRNA and protein. Furthermore, 4′-bromo-resveratrol treatment to melanoma cells resulted in 1) significant induction of apoptosis in melanoma cells, as evident from increase in PARP cleavage, decrease in pro-caspase-3, pro-caspase-8; and 2) G0/G1 phase cell cycle arrest. We also observed a marked increase in WAF-1/p21 protein levels and decreases in cyclin D1 and cdk2 protein levels as a result of 4′-bromo-resveratrol treatment in melanoma cells. Finally, we also found that 4′-bromo-resveratrol caused a metabolic reprograming in human melanoma cells as shown by decreases in the levels of lactate production, glucose uptake, NAD+/NADH ratio, which were accompanied by downregulation in protein levels of lactate dehydrogenase A (LDHA) and glucose transporter 1 (Glut1) in melanoma cells. Collectively, our data suggest that the small molecule SIRT3 inhibitors, including 4′-bromo-resveratrol, may be developed for melanoma management. However, detailed mechanistic studies as well as in vivo validation studies in appropriate animal model(s) are needed to ascertain the clinical potential of 4′-bromo-resveratrol and/or other SIRT3 inhibitors. Citation Format: Jasmine George, Minakshi Nihal, Chandra K. Singh, Nihal Ahmad. Small molecule SIRT3 inhibitor 4′-bromo-resveratrol inhibits proliferation, promotes apoptosis and causes metabolic reprograming of human melanoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1127. doi:10.1158/1538-7445.AM2015-1127

Influence of mild hypothermia therapy on cerebrospinal fluid S-100B and lactate dehydrogenase protein levels and prognosis in acute severe brain injury patients

Objective To observe the effect of mild hypothermia on cerebrospinal fluid S-100B and lactate dehydrogenase (LDH) protein levels and prognosis in acute severe brain injury patients,and to explore their role in treatment of acute severe brain injury.Methods A total of 80 patients with acute severe brain injuries were divided into mild hypothermia group and control group.The patients in mild hypothermia group were treated with hypothermia besides routine treatment,with maintenance of rectal temperature at 33-35 ℃ for 2-7 days.S-100B and LDH protein levels were measured in all patients before and at 1st,3rd and 5th day after the treatment.Prognosis was evaluated with Glasgow Outcome Score (GOS) at 3rd month.Results S-100B and LDH protein levels in the mild hypothermia group were significantly lower than those in the control group after the treatment.GOS scores in the treatment group were higher in the control group at 3rd month (P ＜ 0.05).Conclusion Early use of mild hypothermia can decrease S-100B and LDH protein levels in cerebrospinal fluid and improve prognosis. Key words: Craniocerebral trauma; Hypothermia; S100 proteins; Lactate dehydrogenase proteins ; Prognosis

Docosahexaenoic acid attenuates breast cancer cell metabolism and the Warburg phenotype by targeting bioenergetic function.

Docosahexaenoic acid (DHA; C22:6n-3) depresses mammary carcinoma proliferation and growth in cell culture and in animal models. The current study explored the role of interrupting bioenergetic pathways in BT-474 and MDA-MB-231 breast cancer cell lines representing respiratory and glycolytic phenotypes, respectively and comparing the impacts of DHA with a non-transformed cell line, MCF-10A. Metabolic investigation revealed that DHA supplementation significantly diminished the bioenergetic profile of the malignant cell lines in a dose-dependent manner. DHA enrichment also resulted in decreases in hypoxia-inducible factor (HIF-1α) total protein level and transcriptional activity in the malignant cell lines but not in the non-transformed cell line. Downstream targets of HIF-1α, including glucose transporter 1 (GLUT 1) and lactate dehydrogenase (LDH), were decreased by DHA treatment in the BT-474 cell line, as well as decreases in LDH protein level in the MDA-MB-231 cell line. Glucose uptake, total glucose oxidation, glycolytic metabolism, and lactate production were significantly decreased in response to DHA supplementation; thereby enhancing metabolic injury and decreasing oxidative metabolism. The DHA-induced metabolic changes led to a marked decrease of intracellular ATP levels by 50% in both cancer cell lines, which mediated phosphorylation of metabolic stress marker, AMPK, at Thr172. These findings show that DHA contributes to impaired cancer cell growth and survival by altering cancer cell metabolism, increasing metabolic stress and altering HIF-1α-associated metabolism, while not affecting non-transformed MCF-10A cells. This study provides rationale for enhancement of current cancer prevention models and current therapies by combining them with dietary sources, like DHA.

Exposure to 2,4-dichlorophenoxyacetic acid alters glucose metabolism in immature rat Sertoli cells

The purpose of this study was to determine the effects of 2,4-D, an herbicide used worldwide also known as endocrine disruptor, in Sertoli cell (SC) metabolism.Immature rat SCs were maintained 50h under basal conditions or exposed to 2,4-D (100nM, 10μM and 1mM). SCs exposed to 10μM and 1mM of 2,4-D presented lower intracellular glucose and lactate content. Exposure to 10μM of 2,4-D induced a significant decrease in glucose transporter-3 mRNA levels and phosphofructokinase-1 mRNA levels decreased in cells exposed to 100nM and 10μM of 2,4-D. Exposure to 100nM and 10μM also induced a decrease in lactate dehydrogenase (LDH) mRNA levels while the LDH protein levels were only decreased in cells exposed to 1mM of 2,4-D.Exposure to 2,4-D altered glucose uptake and metabolization in SCs, as well as lactate metabolism and export that may result in impaired spermatogenesis.

Androgen-responsive and nonresponsive prostate cancer cells present a distinct glycolytic metabolism profile

Prostate cancer (PCa) progresses from an early stage, confined to prostate, to a more aggressive metastasized cancer related with loss of androgen responsiveness. Although, it has been recognized that PCa cells have unique metabolic features, their glycolytic profile in androgen-dependent and androgen-independent stages of disease is much less known. Hence, the main purpose of this study was to compare glucose metabolism in androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) PCa cells. Cell culture medium was collected and differences in glucose consumption and, lactate and alanine production were measured using Proton Nuclear Magnetic Resonance (1H NMR) spectra analysis. The mRNA and protein expression of glucose transporters (GLUT1 and GLUT3), phosphofructokinase 1 (PFK1), lactate dehydrogenase (LDH) and monocarboxylate transporter (MCT4) were determined by real-time PCR and Western Blot, respectively. The obtained results demonstrate that androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) cells consumed similar amounts of glucose, whereas PC3 cells present higher lactate production. This increase in lactate production was concomitant with higher levels of MCT4 protein, increased LDH activity and higher lactate/alanine ratio, also suggesting increased levels of oxidative stress in PC3 cells. However, protein levels of LDH, associated with lactate metabolism, and GLUT3, involved in glucose uptake, were decreased in PC3 comparatively with LNCaP. Androgen-responsive and nonresponsive PCa cells present distinct glycolytic metabolism profiles, which suggest that targeting LDH and MCT4 metabolic pathways may be an important step for the development of new diagnostic and therapeutic strategies in the different stages of PCa.

Purification and Properties of Hypoxically Induced Lactate Dehydrogenase from Barley Roots

Using Affigel Blue and oxamate-agarose affinity chromatography, lactate dehydrogenase (LDH) was purified 2000-fold from hypoxically induced barley roots. Molecular weights of the native and sodium dodecyl sulfate-denatured LDH protein were 157 and 40 kilodaltons, respectively, indicating a tetramer. Purified barley LDH was very similar in size and kinetic properties to potato LDH. However, their amino acid compositions differed substantially and antibodies raised against barley LDH did not cross-react with potato LDH on immunoblots, implying that the barley and potato LDHs are not closely related proteins. In vivo [(35)S] methionine labeling and immunoprecipitation experiments indicated that hypoxia increased the rate of LDH protein synthesis, and immunoblot analysis showed that LDH protein levels rose during hypoxia. We conclude that increased enzyme synthesis plays a major part in the induction of LDH enzyme activity by low O(2) levels in barley roots.