ANXA2P2/miR-9/LDHA axis regulates Warburg effect and affects glioblastoma proliferation and apoptosis

BackgroundAerobic glycolysis is a unique tumor cell phenotype considered as one of the hallmarks of cancer. Aerobic glycolysis can accelerate tumor development by increasing glucose uptake and lactate production. In the present study, lactate dehydrogenase A (LDHA) is significantly increased within glioma tissue samples and cells, further confirming the oncogenic role of LDHA within glioma.MethodsHematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were applied for histopathological examination. The protein levels of LDHA, transporter isoform 1 (GLUT1), hexokinase 2 (HK2), phosphofructokinase (PFK) in target cells were detected by Immunoblotting. The predicted miR-9 binding to lncRNA Annexin A2 Pseudogene 2 (ANXA2P2) or the 3′ untranslated region (UTR) of LDHA was verified using Luciferase reporter assay. Cell viability or apoptosis were examined by MTT assay or Flow cytometry. Intracellular glucose and Lactate levels were measured using glucose assay kit and lactate colorimetric assay kit.ResultsThe expression of ANXA2P2 showed to be dramatically upregulated within glioma tissue samples and cells. Knocking down ANXA2P2 within glioma cells significantly inhibited cell proliferation and aerobic glycolysis, as manifested as decreased lactate and increased glucose in culture medium, and downregulated protein levels of glycolysis markers, GLUT1, HK2, PFK, as well as LDHA. miR-9 was predicted to target both lncRNA ANXA2P2 and LDHA. The overexpression of miR-9 suppressed the cell proliferation and aerobic glycolysis of glioma cells. Notably, miR-9 could directly bind to LDHA 3′UTR to inhibit LDHA expression and decrease the protein levels of LDHA. ANXA2P2 competitively targeted miR-9, therefore counteracting miR-9-mediated repression on LDHA. Within tissues, miR-9 exhibited a negative correlation with ANXA2P2 and LDHA, respectively, whereas ANXA2P2 and LDHA exhibited a positive correlation with each other.ConclusionsIn conclusion, ANXA2P2/miR-9/LDHA axis modulates the aerobic glycolysis progression in glioma cells, therefore affecting glioma cell proliferation.     

AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: Implication of the cell survival in mitochondrial diseases

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H₂O₂. The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H₂O₂-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H₂O₂ in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H₂O₂ was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H₂O₂-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H₂O₂-treated normal skin fibroblasts to oxidative stress.     

Glucose transporter GLUT1 expression is important for oriental river prawn (Macrobrachium nipponense) hemocyte adaptation to hypoxic conditions

Crustaceans have an open vascular system in which hemocytes freely circulate in hemolymph. Hemocytes are rich in hemocyanin, a specific oxygen-transport protein in crustaceans; therefore, understanding the response of hemocytes to hypoxia is crucial. Although hemocytes take up glucose during hypoxia, the molecular mechanism of glucose uptake in crustaceans remains unclear. Herein, we identified two highly conserved glucose transporters (GLUT1 and GLUT2) in Macrobrachium nipponense (oriental river prawn) and analyzed their tissue-specific expression patterns. Our immunofluorescence assays showed that GLUT1 and GLUT2 are located on the cell membrane, with a strong GLUT1 signal in primary hemocytes under hypoxia. We found that during acute hypoxia, hypoxia-inducible factor-1α–related metabolic alterations result in decreased mitochondrial cytochrome c oxidase activity, implying a classic glycolytic mechanism. As a proof of concept, we replicated these findings in insect S2 cells. Acute hypoxia significantly induced hypoxia-inducible factor-1α, GLUT1, and pyruvate dehydrogenase kinase isozyme 1 expression in primary hemocytes, and hypoxia-induced increases in glucose uptake and lactate secretion were observed. GLUT1 knockdown induced intracellular reactive oxygen species generation and apoptosis in vitro and in vivo, resulting in increased prawn mortality and more apoptotic cells in their brains, implying a vital function of GLUT1 in hypoxia adaptation. Taken together, our results suggest a close relationship between hypoxia-mediated glycolysis and GLUT1 in hemocytes. These results demonstrated that in crustaceans, adaptation to hypoxia involves glucose metabolic plasticity.     

Quadriceps metabolism during constant workrate cycling exercise in chronic obstructive pulmonary disease

Impaired resting metabolism in peripheral muscles potentially contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). This study investigated the cytosolic energy metabolism of the quadriceps, from glycogen degradation to lactate accumulation, in exercising patients with COPD, in comparison to healthy controls. We measured, in 12 patients with COPD and 10 control subjects, resting and post-cycling exercise quadriceps levels of 1) energy substrates and end products of glycolysis (glycogen, glucose, pyruvate, and lactate) and intermediate markers of glycolysis (glucose-6-phosphate, glucose-1-phosphate, fructose-6-phosphate) and 2) the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase, lactate dehydrogenase). Exercise intensity (P < 0.01), duration (P = 0.049), and total work (P < 0.01) were reduced in patients with COPD. The variations in energy substrates and end products of glycolysis after cycling exercise were of similar magnitude in patients with COPD and controls. Glucose-6-phosphate (P = 0.036) and fructose-6-phosphate (P = 0.042) were significantly elevated in patients with COPD after exercise. Phosphofructokinase (P < 0.01) and lactate dehydrogenase (P = 0.02) activities were greater in COPD. Muscle glycogen utilization (P = 0.022) and lactate accumulation (P = 0.025) per unit of work were greater in COPD. We conclude that cycling exercise induced changes in quadriceps metabolism in patients with COPD that were of similar magnitude to those of healthy controls. These intramuscular events required a much lower exercise work load and time to occur in COPD. Our data suggest a greater reliance on glycolysis during exercise in COPD, which may contribute to exercise intolerance in COPD.     

Changes in gene expression involved in energy utilization during chicken follicle development

Ovarian follicle development in egg-laying species is characterized by rapid growth in 7 days prior to ovulation when DNA and protein synthesis is markedly increased in the granulosa and theca cells. However, energy and substrate sources to facilitate the extensive DNA and protein synthesis necessary for folliculogenesis have not been identified in avian species. The current study was undertaken to investigate the expression profiles of regulatory genes involved in glucose transport, glycolysis and fatty acid oxidation in the follicle membranes from the small white follicle (SWF) to follicle 1 (F1) stages of follicle development. In our analysis of glucose transporter (GLUT) isoform expression, the level of GLUT1 mRNA increased with follicle development while GLUT2, GLUT3 and GLUT8 mRNA levels were unaffected by follicle development. In contrast, the expression patterns of proteins involved in metabolism down-stream of glucose transport, including hexokinase (HK), pyruvate dehydrogenase E1alpha (PDH E1alpha) and citrate synthase (CS), did not vary with the developmental stage of the follicle, even during rapid follicle growth. Expression of genes related to beta-oxidation of fatty acids (carnitine palmityl CoA transferase I and II, l-3-hydroxyacyl CoA dehydrogenase and long-chain acyl-CoA dehydrogenase), for which expression in the ovarian follicles of mammalian species has not previously been studied, was not changed consistently with the follicle development. These results suggest that both glucose and fatty acids might work as energy sources to ensure rapid follicle development in the chicken ovary, even though glycolysis and beta-oxidation are not modulated by follicle development.     

Modulation of glucose transporter 1 (GLUT1) expression levels alters mouse mammary tumor cell growth in vitro and in vivo

Tumor cells exhibit an altered metabolism characterized by elevated aerobic glycolysis and lactate secretion which is supported by an increase in glucose transport and consumption. We hypothesized that reducing or eliminating the expression of the most prominently expressed glucose transporter(s) would decrease the amount of glucose available to breast cancer cells thereby decreasing their metabolic capacity and proliferative potential.Of the 12 GLUT family glucose transporters expressed in mice, GLUT1 was the most abundantly expressed at the RNA level in the mouse mammary tumors from MMTV-c-ErbB2 mice and cell lines examined. Reducing GLUT1 expression in mouse mammary tumor cell lines using shRNA or Cre/Lox technology reduced glucose transport, glucose consumption, lactate secretion and lipid synthesis in vitro without altering the concentration of ATP, as well as reduced growth on plastic and in soft agar. The growth of tumor cells with reduced GLUT1 expression was impaired when transplanted into the mammary fat pad of athymic nude mice in vivo. Overexpression of GLUT1 in a cell line with low levels of endogenous GLUT1 increased glucose transport in vitro and enhanced growth in nude mice in vivo as compared to the control cells with very low levels of GLUT1.These studies demonstrate that GLUT1 is the major glucose transporter in mouse mammary carcinoma models overexpressing ErbB2 or PyVMT and that modulation of the level of GLUT1 has an effect upon the growth of mouse mammary tumor cell lines in vivo.     

Pioglitazone increases the glycolytic efficiency of human Sertoli cells with possible implications for spermatogenesis

Pioglitazone is a synthetic agonist for the nuclear receptor peroxisome proliferator-activated receptor γ used to treat type 2 diabetes mellitus. Recently we reported that antidiabetic drugs regulate the nutritional support of spermatogenesis by Sertoli cells. Herein, we investigate the effects of pioglitazone on human Sertoli cells metabolism. Human Sertoli cells were cultured in the presence of pioglitazone (1, 10, 100 μM). Protein levels of phosphofructokinase 1, lactate dehydrogenase, hexokinase, glucose transporters (GLUT1, GLUT2, GLUT3), monocarboxylate transporter 4 and oxidative phosphorylation complexes were determined by Western blot. Lactate dehydrogenase and alanine aminotransferase activity were assessed and metabolite production and consumption determined by proton nuclear magnetic resonance. Mitochondrial membrane potential was also determined. Glucose consumption more than doubled in human Sertoli cells stimulated with pioglitazone 100 μM. Mitochondrial complex II protein levels increased 50% with exposure to pioglitazone (100 μM) in human Sertoli cells, though mitochondrial membrane potential was decreased by 32%. The pharmacological concentration of pioglitazone (10 μM) almost doubled lactate production and established crucial correlations among key intervenient of glycolysis. Moreover, in the same concentration, alanine aminotransferase decreased more than 80%. Our results suggest that pioglitazone (10 μM) increases the efficiency of the glycolytic flux and lactate production by human Sertoli cells, which is essential to sustain and preserve the spermatogenic event. Thus, pioglitazone may improve male fertility and thus, be considered a suitable antidiabetic drug for men in reproductive age.     

Inhibition of eIF5A hypusination reprogrammes metabolism and glucose handling in mouse kidney

Inhibition of the eukaryotic initiation factor 5A activation by the spermidine analogue GC7 has been shown to protect proximal cells and whole kidneys against an acute episode of ischaemia. The highlighted mechanism involves a metabolic switch from oxidative phosphorylation toward glycolysis allowing cells to be transiently independent of oxygen supply. Here we show that GC7 decreases protein expression of the renal GLUT1 glucose transporter leading to a decrease in transcellular glucose flux. At the same time, GC7 modifies the native energy source of the proximal cells from glutamine toward glucose use. Thus, GC7 acutely and reversibly reprogrammes function and metabolism of kidney cells to make glucose its single substrate, and thus allowing cells to be oxygen independent through anaerobic glycolysis. The physiological consequences are an increase in the renal excretion of glucose and lactate reflecting a decrease in glucose reabsorption and an increased glycolysis. Such a reversible reprogramming of glucose handling and oxygen dependence of kidney cells by GC7 represents a pharmacological opportunity in ischaemic as well as hyperglycaemia-associated pathologies from renal origin.Subject terms: Cell biology, Physiology     

Carbohydrate metabolism in the rat peritoneal macrophages

Rat peritoneal macrophages derive energy differently from other tissues. Resting rat peritoneal macrophages have been taken for the present investigation. Lactate produced by extracellular glycolysis in the peritoneal lavage fluid, is readily converted into pyruvate by resting peritoneal macrophages and is oxidised in mitochondria. Glycolytic enzymes other than phosphoglucoisomerase and lactate dehydrogenase could not be substantially demonstrated. Glucose-6-phosphate dehydrogenase was detected. The presence of glucose-6-phosphate dehydrogenase along with phosphoglucoisomerase indicates the operation of the hexose monophosphate shunt as a pathway supplementary to glycolysis. Resting rat peritoneal macrophages thus appear to utilize extracellular lactate as their main energy source instead of glucose, bypass glycolysis and have active hexose monophosphate shunt.     

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 ((1)H 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.     

Crosstalk of mTOR/PKM2 and STAT3/c-Myc signaling pathways regulate the energy metabolism and acidic microenvironment of gastric cancer

Cancer cells consume large amounts of glucose to produce lactate, even in the presence of ample oxygen. This phenomenon is called the Warburg effect. c-Myc is an important member of the Myc gene family and is involved in the development of various tumors. It plays an important role in the regulation of tumor energy metabolism, which can regulate glycolysis to promote the Warburg effect in a tumor. Our study aimed to improve the malignant biological behavior by controlling the energy metabolism of gastric cancer through the mTOR/PKM2 and signal transduction and activator 3 (STAT3)/c-Myc signaling pathways through a series of in vitro experiments. Human gastric cancer AGS and HGC-27 cells were treated with PKM2 and c-Myc lentivirus, and the effects of the knockdown of PKM2 and/or c-Myc were analyzed on cell proliferation, cell apoptosis, the ability of cell migration, and the growth signaling pathway in vitro. The expressions of PKM2, c-Myc, LDHA, STAT3, P-STAT3, GLUT-1 gene were identified by the quantitative real-time polymerase chain reaction and Western blot analysis. Lactate and glucose levels were tested by the corresponding kit. Our findings showed that PKM2 and c-Myc were upregulated in human gastric cancer. Knockdown of c-Myc in gastric cancer cells suppressed cell proliferation capacity and glycolysis level, and the inhibitory effects on gastric cancer cells upon co-knockdown of PKM2 and c-Myc were more obvious compared with knockout of PKM2 or c-Myc alone. And there was a correlation between the mTOR/PKM2 and the STAT3/c-Myc signaling pathways. Our results suggested that c-Myc might be considered a potential therapeutic target for gastric cancer and PKM2 combined with c-Myc could better inhibit the malignant biological behaviors of gastric cancer.