ATP produced by anaerobic glycolysis is essential for enucleation of human erythroblasts
2019; Elsevier BV; Volume: 72; Linguagem: Inglês
10.1016/j.exphem.2019.02.004
ISSN1873-2399
AutoresTatsufumi Goto, Kumi Ubukawa, Isuzu Kobayashi, Kotomi Sugawara, Ken Asanuma, Yumi Sasaki, Yong‐Mei Guo, Naoto Takahashi, Kenichi Sawada, Hideki Wakui, Wataru Nunomura,
Tópico(s)Cancer, Hypoxia, and Metabolism
Resumo•Low O2 conditions accelerated human CD34+ cell erythroblast differentiation.•Inhibition of key enzymes in anaerobic glycolysis blocked erythroblast enucleation.•Inhibition of key enzymes in anaerobic glycolysis reduced ATP levels in erythroblasts.•Phosphorylation of PDH constituted an important event for erythroblast differentiation.•Sustained expression of MCT1 was observed during erythroblast differentiation. More than 2million human erythroblasts extrude their nuclei every second in bone marrow under hypoxic conditions (<7% O2). Enucleation requires specific signal transduction pathways and the local assembly of contractile actomyosin rings. However, the energy source driving these events has not yet been identified. We examined whether different O2 environments (hypoxic [5% O2] and normoxic [21% O2] conditions) affected human CD34+ cell erythroblast differentiation. We also investigated the regulatory mechanisms underlying energy production in erythroblasts during terminal differentiation under 5% or 21% O2 conditions. The results obtained revealed that the enucleation ratio and intracellular levels of adenosine triphosphate (ATP), lactate dehydrogenase (LDH) M3H, and hypoxia-inducible factor 1α in erythroblasts during terminal differentiation were higher under the 5% O2 condition than under the 21% O2 condition. We also found that the enzymatic inhibition of glyceraldehyde 3-phosphate dehydrogenase and LDH, key enzymes in anaerobic glycolysis, blocked the proliferation of colony-forming units–erythroid and enucleation of erythroblasts, and also reduced ATP levels in erythroblasts under both hypoxic and normoxic conditions. Under both conditions, phosphorylation of the Ser232, Ser293, and Ser300 residues in pyruvate dehydrogenase (inactive state of the enzyme) in erythroblasts was involved in regulating the pathway governing energy metabolism during erythroid terminal differentiation. This reaction may be mediated by pyruvate dehydrogenase kinase (PDK) 4, the major PDK isozyme expressed in erythroblasts undergoing enucleation. Collectively, these results suggest that ATP produced by anaerobic glycolysis is the main source of energy for human erythroblast enucleation in the hypoxic bone marrow environment. More than 2million human erythroblasts extrude their nuclei every second in bone marrow under hypoxic conditions (<7% O2). Enucleation requires specific signal transduction pathways and the local assembly of contractile actomyosin rings. However, the energy source driving these events has not yet been identified. We examined whether different O2 environments (hypoxic [5% O2] and normoxic [21% O2] conditions) affected human CD34+ cell erythroblast differentiation. We also investigated the regulatory mechanisms underlying energy production in erythroblasts during terminal differentiation under 5% or 21% O2 conditions. The results obtained revealed that the enucleation ratio and intracellular levels of adenosine triphosphate (ATP), lactate dehydrogenase (LDH) M3H, and hypoxia-inducible factor 1α in erythroblasts during terminal differentiation were higher under the 5% O2 condition than under the 21% O2 condition. We also found that the enzymatic inhibition of glyceraldehyde 3-phosphate dehydrogenase and LDH, key enzymes in anaerobic glycolysis, blocked the proliferation of colony-forming units–erythroid and enucleation of erythroblasts, and also reduced ATP levels in erythroblasts under both hypoxic and normoxic conditions. Under both conditions, phosphorylation of the Ser232, Ser293, and Ser300 residues in pyruvate dehydrogenase (inactive state of the enzyme) in erythroblasts was involved in regulating the pathway governing energy metabolism during erythroid terminal differentiation. This reaction may be mediated by pyruvate dehydrogenase kinase (PDK) 4, the major PDK isozyme expressed in erythroblasts undergoing enucleation. Collectively, these results suggest that ATP produced by anaerobic glycolysis is the main source of energy for human erythroblast enucleation in the hypoxic bone marrow environment. Mammalian erythropoiesis culminates in enucleation, a still partially understood process that entails the expulsion of the nucleus from the cytoplasm of erythroblasts. During erythropoiesis, stem cells undergo lineage-specific commitment and generate erythroid progenitor cells through cellular division events, which include nuclear (mitosis) and cytoplasmic (cytokinesis) components. These progenitor cells consist of burst-forming units–erythroid (BFU-Es) and their progeny, colony-forming units–erythroid (CFU-Es) [1Sawada K Krantz SB Kans JS et al.Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.J Clin Invest. 1987; 80: 357-366Crossref PubMed Scopus (166) Google Scholar, 2Sawada K Krantz SB Dai CH et al.Transitional change of colony stimulating factor requirements for erythroid progenitors.J Cell Physiol. 1991; 149: 1-8Crossref PubMed Scopus (32) Google Scholar]. Over the course of an additional 6 to 7days, human CFU-Es proliferate and differentiate into mature erythroblasts [3Hebiguchi M Hirokawa M Guo YM et al.Dynamics of human erythroblast enucleation.Int J Hematol. 2008; 88: 498-507Crossref PubMed Scopus (31) Google Scholar, 4Ubukawa K Guo YM Takahashi M et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (61) Google Scholar, 5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Mammalian erythroblasts then undergo enucleation, become reticulocytes, and, finally, mature erythrocytes. Expelled nuclei are phagocytosed by macrophages [6Chasis JA Mohandas N Erythroblastic islands: niches for erythropoiesis.Blood. 2008; 112: 470-478Crossref PubMed Scopus (343) Google Scholar]. More than 2million erythroblasts extrude their nuclei every second [7Sackmann E Biological membranes architecture and function.in: Lipowsky R Sackmann E Handbook of Biological Physics. 1. Elsevier Science, Amsterdam1995: 1-62Google Scholar]. The process of enucleation may be similar to cytokinesis, and many of the general principles of cytokinesis apply to enucleation. In cytokinesis and enucleation, the cytoskeleton plays a key role in the positioning of the division site. Once this site is selected, the local assembly of contractile actomyosin rings occurs, during which nonmuscle myosin IIB remodels the plasma membrane [4Ubukawa K Guo YM Takahashi M et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (61) Google Scholar]. Trafficking of the necessary components to the division site and membrane fusion lead to the physical separation of daughter cells [8Glotzer M. The molecular requirements for cytokinesis.Science. 2005; 307: 1735-1739Crossref PubMed Scopus (561) Google Scholar, 9Barr FA Gruneberg U Cytokinesis: placing and making the final cut.Cell. 2007; 131: 847-860Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar]. Moreover, several key signal transduction effectors, such as phosphoinositide 3-kinase [10Wang J Ramirez T Ji P Jayapal SR Lodish HF Murata-Hori M Mammalian erythroblast enucleation requires PI3K-dependent cell polarization.J Cell Sci. 2012; 125: 340-349Crossref PubMed Scopus (41) Google Scholar], contribute to the enucleation process mainly through phosphorylation events. These findings strongly support enucleation requiring adenosine triphosphate (ATP) as an energy source. The concentration of oxygen in the bone marrow environment, in which erythroblasts develop, differentiate, and extrude their nucleus, is less than 7% [11Spencer JA Ferraro F Roussakis E et al.Direct measurement of local oxygen concentration in the bone marrow of live animals.Nature. 2014; 508: 269-273Crossref PubMed Scopus (689) Google Scholar, 12Nombela-Arrieta C Pivarnik G Winkel B et al.Quantitative imaging of hematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment.Nat Cell Biol. 2013; 15: 533-543Crossref PubMed Scopus (369) Google Scholar]. This feature implies that erythroblasts extrude their nuclei under hypoxic conditions and suggests that the ATP necessary for enucleation is produced by anaerobic glycolysis rather than by oxidative phosphorylation in mitochondria. This assumption appears to be supported by the following observations on other processes that occur in the hypoxic bone marrow environment: Simsek et al. [13Simsek T Kocabas F Zheng J et al.The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche.Cell Stem Cell. 2010; 7: 380-390Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar] and Kocabas et al. [14Kocabas F Xie L Xie J et al.Hypoxic metabolism in human hematopoietic stem cells.Cell Biosci. 2015; 5: 39Crossref PubMed Scopus (46) Google Scholar] reported that mouse and human long-term hematopoietic stem cells (LT-HSCs) use glycolysis instead of mitochondrial oxidative phosphorylation as their main energy source. These unique metabolic properties of LT-HSCs are considered to depend on hypoxia-inducible factor (HIF)-1α–driven metabolic pathways [13Simsek T Kocabas F Zheng J et al.The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche.Cell Stem Cell. 2010; 7: 380-390Abstract Full Text Full Text PDF PubMed Scopus (724) Google Scholar, 14Kocabas F Xie L Xie J et al.Hypoxic metabolism in human hematopoietic stem cells.Cell Biosci. 2015; 5: 39Crossref PubMed Scopus (46) Google Scholar]. Glycolysis is a series of metabolic processes by which one molecule of glucose is catabolized to two molecules of pyruvate with a net gain of two ATP [15Pelicano H Martin DS Xu RH Huang P Glycolysis inhibition for anticancer treatment.Oncogene. 2006; 25: 4633-4646Crossref PubMed Scopus (1083) Google Scholar]. Under anaerobic conditions, glycolysis is the main energy source in living cells, and nicotinamide adenine dinucleotide (NAD+ [oxidized form]), which is essential for the catalytic reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is regenerated from NADH (reduced form) by the reduction of pyruvate to lactate catalyzed by lactate dehydrogenase (LDH) [15Pelicano H Martin DS Xu RH Huang P Glycolysis inhibition for anticancer treatment.Oncogene. 2006; 25: 4633-4646Crossref PubMed Scopus (1083) Google Scholar]. In anaerobic glycolysis, monocarboxylate transporters (MCTs), in which MCT1 is ubiquitously expressed, are essential for the transport of increased lactate across the plasma membranes [16Halestrap AP Price NT. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.Biochem J. 1999; 343: 281-299Crossref PubMed Scopus (1046) Google Scholar]. To generate ATP through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation in the mitochondria, dephosphorylated pyruvate dehydrogenase (PDH) catalyzes the irreversible oxidative decarboxylation of pyruvate into acetyl-coenzyme A (CoA) [15Pelicano H Martin DS Xu RH Huang P Glycolysis inhibition for anticancer treatment.Oncogene. 2006; 25: 4633-4646Crossref PubMed Scopus (1083) Google Scholar, 17Adeva M González-Lucán M Seco M Donapetry C Enzymes involved in L-lactate metabolism in humans.Mitochondrion. 2013; 13: 615-629Crossref PubMed Scopus (53) Google Scholar]. The phosphorylation of PDH is accomplished by PDH kinases (PDKs), which consequently inactivate the enzymatic activity of PDH [17Adeva M González-Lucán M Seco M Donapetry C Enzymes involved in L-lactate metabolism in humans.Mitochondrion. 2013; 13: 615-629Crossref PubMed Scopus (53) Google Scholar]. Three phosphorylation sites have been identified on the α subunit of human PDH: Ser232, Ser293, and Ser300 [18Rardin MJ Wiley SE Naviaux RK Murphy AN Dixon JE Monitoring phosphorylation of the pyruvate dehydrogenase complex.Anal Biochem. 2009; 389: 157-164Crossref PubMed Scopus (103) Google Scholar]. The superfamily of glucose transporters (GLUTs) comprises 14 isoforms in the human genome. Among them, GLUT1 is the main functional transporter in various hematopoietic cell lineages, including erythroblasts and mature erythrocytes [19Montel-Hagen A Blanc L Boyer-Clavel M et al.The Glut1 and Glut4 glucose transporters are differentially expressed during perinatal and postnatal erythropoiesis.Blood. 2008; 112: 4729-4738Crossref PubMed Scopus (57) Google Scholar, 20Montel-Hagen A Sitbon M Taylor N Erythroid glucose transporters.Curr Opin Hematol. 2009; 16: 165-172Crossref PubMed Scopus (45) Google Scholar]. However, limited information is currently available on the regulation and function of GLUT1 during erythropoiesis. According to the findings reported by Montel-Hagen etal. [21Montel-Hagen A Kinet S Manel N et al.Erythrocyte Glut1 triggers dehydroascorbic acid uptake in mammals unable to synthesize vitamin C.Cell. 2008; 132: 1039-1048Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar], glucose transport actually decreases during human erythropoiesis despite an increase in GLUT1 transcripts, which naturally raises the question of the nature and regulation of energy metabolism in human erythroblasts. Limited information is currently available on ex vivo models of the regulation of erythropoiesis by low O2 concentrations [22Vlaski M Lafarge X Chevaleyre J Duchez P Boiron JM Ivanovic Z Low oxygen concentration as a general physiologic regulator of erythropoiesis beyond the EPO-related downstream tuning and a tool for the optimization of red blood cell production ex vivo.Exp Hematol. 2009; 37: 573-584Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. In the present study, we examined whether different O2 environments (hypoxic [5% O2] and normoxic [21% O2] conditions) affect human CD34+ cell erythroblast differentiation. We also investigated the regulatory mechanisms underlying energy production in erythroblasts during terminal differentiation under hypoxic or normoxic conditions. The results obtained revealed the acceleration of erythroblast enucleation under hypoxic conditions and led us to propose mechanisms for the production of ATP via anaerobic glycolysis during the terminal differentiation of erythroblasts including enucleation. Bovine serum albumin (BSA) and Iscove's Modified Dulbecco's Medium (IMDM) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI-1640 medium was from Life Technologies (Carlsbad, CA). Fetal calf serum (FCS) was from Flow Laboratories (McLean, VA) and Hyclone Laboratories (Logan, UT). Penicillin and streptomycin were from Flow Laboratories. Insulin was from Wako Pure Chemical Industries (Osaka, Japan). Interleukin-3 (IL-3) and stem cell factor (SCF) were gifts from Kirin Brewery (Tokyo, Japan). Erythropoietin (EPO) was from Chugai Pharmaceutical (Tokyo, Japan). Vitamin B12 was from Eisai (Tokyo, Japan), and folic acid was from Takeda Pharmaceutical (Osaka, Japan). The GAPDH inhibitor koningic acid (KA) [23Kumagai S Narasaki R Hasumi K Glucose-dependent active ATP depletion by koningic acid kills high-glycolytic cells.Biochem Biophys Res Commun. 2008; 365: 362-368Crossref PubMed Scopus (40) Google Scholar] was from Adipogen (San Diego, CA). The LDH inhibitor stiripentol (STP) [24Sada N Lee S Katsu T Otsuki T Inoue T Targeting LDH enzymes with a stiripentol analog to treat epilepsy.Science. 2015; 347: 1362-1367Crossref PubMed Scopus (220) Google Scholar] was from Tokyo Kasei (Tokyo, Japan). Granulocyte colony-stimulating factor (G-CSF)–mobilized human peripheral blood CD34+ cells were purified as previously described [25Oda A Sawada K Druker BJ et al.Erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B in primary cultured human erythroid precursors.Blood. 1998; 92: 443-451Crossref PubMed Google Scholar] and stored in liquid nitrogen until further use. Informed consent was obtained from all participants prior to their entry into this study, which was approved by the Akita University Graduate School of Medicine Committee for the Protection of Human Subjects. To generate erythroid progenitor cells, CD34+ cells prepared from the same individual were thawed and cultured as previously described [3Hebiguchi M Hirokawa M Guo YM et al.Dynamics of human erythroblast enucleation.Int J Hematol. 2008; 88: 498-507Crossref PubMed Scopus (31) Google Scholar, 4Ubukawa K Guo YM Takahashi M et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (61) Google Scholar, 5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Briefly, cells were cultured in phase I medium (IMDM erythroid medium containing 20% FCS, 10% heat-inactivated pooled human AB serum, 1% BSA, 10 μg/mL insulin, 0.5 μg/mL vitamin B12, 15 μg/mL folic acid, 50nmol/L β-mercaptoethanol, 50U/mL penicillin, 50 μg/mL streptomycin in the presence of 50ng/mL IL-3, 50ng/mL SCF, and 2IU/mL EPO). Cells were maintained in a 5% CO2 incubator (MCO-170AICUVH, PHC Holdings, Tokyo, Japan) at 37°C under 21% O2 conditions or in a Multi-Gas Incubator (MCO-5M, PHC Holdings) at 37°C under 5% CO2 and 5% O2 conditions. After 7days in culture, cells were harvested and washed three times with IMDM containing 0.3% BSA. The maturation stage of day 7 cells was similar to that of CFU-Es [3Hebiguchi M Hirokawa M Guo YM et al.Dynamics of human erythroblast enucleation.Int J Hematol. 2008; 88: 498-507Crossref PubMed Scopus (31) Google Scholar, 4Ubukawa K Guo YM Takahashi M et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (61) Google Scholar, 5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Hereafter, day 7 cells were referred to as CFU-Es. Aliquots of CFU-Es (1 × 105 cells) were then cultured in phase II medium (erythroid medium with EPO alone, without β-mercaptoethanol, IL-3, or SCF), as previously described [3Hebiguchi M Hirokawa M Guo YM et al.Dynamics of human erythroblast enucleation.Int J Hematol. 2008; 88: 498-507Crossref PubMed Scopus (31) Google Scholar, 4Ubukawa K Guo YM Takahashi M et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (61) Google Scholar, 5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar] under 5% or 21% O2 conditions, with or without the inhibitors KA and STP dissolved in dimethyl sulfoxide (DMSO). Enucleation was evaluated as previously described [4Ubukawa K Guo YM Takahashi M et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (61) Google Scholar, 5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Briefly, cultured cells were spun onto slides using Cytospin 3 (Shandon Lipshaw, Pittsburgh, PA) and were stained with May–Grünwald–Giemsa reagent. Enucleation was defined as the expulsion of the nucleus to the outside of the reticulocyte. Reticulocytes touching expelled nuclei or with a thin connecting strand between the reticulocyte and nucleus were considered to be the earliest enucleated cells. The enucleation fraction among cytospin cells was similar to that among cells prepared without mechanical force (without centrifugation). The enucleation fraction was calculated using the formula (erythrocytes/[erythrocytes + erythroblasts]) × 100 (%), with 300 cells, including erythrocytes and erythroblasts, for each slide. Yield and viability were assessed based on dye exclusion using 0.2% trypan blue. Triplicate cultures were used at each time point. Flow cytometry was performed as previously described [5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Briefly, cells collected from cultures were washed twice with IMDM containing 0.3% BSA and incubated with a phycoerythrin-conjugated mouse monoclonal antibody to human CD71 (transferrin receptor; BD Biosciences, Franklin Lakes, NJ) and fluorescein isothiocyanate-conjugated mouse monoclonal antibody to human glycophorin A (GPA; Dako, Santa Clara, CA). Cells were then washed twice with 10mmol/L sodium phosphate buffer, pH 7.4, 0.15mol/L NaCl (phosphate-buffered saline [PBS]) containing 0.5% BSA and analyzed using a FACSCanto II (BD Biosciences). Intracellular ATP levels in erythroblasts were measured based on the luciferin–luciferase reaction. Briefly, 1 × 104 cultured cells per sample were resuspended in 100 μL of IMDM and placed in each well of a 96-well plate. Light emission was recorded in triplicate using a luminometer (Tecan Group, Männedorf, Switzerland) after the addition of 100 μL of the "Cellno" ATP assay reagent (TOYO B-Net, Tokyo, Japan). During the terminal differentiation of erythroblasts, 1 × 105 cultured cells were incubated in serum- and glucose-free RPMI-1640 medium for 30min. After serum and glucose starvation, glucose uptake was initiated by the addition of 2-deoxy-D-[1,2-3H]glucose (PerkinElmer, Waltham, MA) to a final concentration of 2.5 μmol/L (74 kBq). Uptake assays were performed for 30min. Cells were then washed twice with PBS and resuspended in 6mL of AQUASOL-II (PerkinElmer Japan, Yokohama, Japan). The uptake of 2-deoxy-D-[1,2-3H]glucose was assessed in triplicate using the liquid scintillation counter LSC-8000 (Hitachi Aloka Medical, Tokyo, Japan). Real-time PCR was performed as previously described [5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. Briefly, total RNA was extracted from 2 × 104 cultured cells per sample using TRIzol reagent (Invitrogen, Carlsbad, CA) or an RNeasy Mini kit (QIAGEN, Hilden, Germany). Extracted RNA was reverse-transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen). The resultant cDNA was then subjected to real-time PCR using LightCycler 480 SYBR Green I Master (Roche Applied Science, Mannheim, Germany). PCR primer sequences for the GLUT1 [26Xu H Zhao L Fang Q et al.MiR-338-3p inhibits hepatocarcinoma cells and sensitizes these cells to sarafenib by targeting hypoxia-induced factor 1α.PLoS One. 2014; 9e115565Crossref PubMed Scopus (81) Google Scholar], HIF1A [26Xu H Zhao L Fang Q et al.MiR-338-3p inhibits hepatocarcinoma cells and sensitizes these cells to sarafenib by targeting hypoxia-induced factor 1α.PLoS One. 2014; 9e115565Crossref PubMed Scopus (81) Google Scholar], MCT1 [27de Heredia FP Wood IS Trayhurn P Hypoxia stimulates lactate release and modulates monocarboxylate transporter (MCT1, MCT2, and MCT4) expression in human adipocytes.Eur J Physiol. 2010; 459: 509-518Crossref PubMed Scopus (108) Google Scholar], PDK1 [28Spriet LL Tunstall RJ Watt MJ Mehan KA Hargreaves M Cameron-Smith D Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting.J Appl Physiol. 2004; 96: 2082-2087Crossref PubMed Scopus (70) Google Scholar], PDK2 [28Spriet LL Tunstall RJ Watt MJ Mehan KA Hargreaves M Cameron-Smith D Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting.J Appl Physiol. 2004; 96: 2082-2087Crossref PubMed Scopus (70) Google Scholar], PDK3 [28Spriet LL Tunstall RJ Watt MJ Mehan KA Hargreaves M Cameron-Smith D Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting.J Appl Physiol. 2004; 96: 2082-2087Crossref PubMed Scopus (70) Google Scholar], and PDK4 [28Spriet LL Tunstall RJ Watt MJ Mehan KA Hargreaves M Cameron-Smith D Pyruvate dehydrogenase activation and kinase expression in human skeletal muscle during fasting.J Appl Physiol. 2004; 96: 2082-2087Crossref PubMed Scopus (70) Google Scholar] genes are described in Table1. Relative gene expression levels were normalized with the 28S rRNA gene as previously described [29Guo YM Ishii K Hirokawa M et al.CpG-ODN 2006 and human parvovirus B19 genome consensus sequences selectively inhibit growth and development of erythroid progenitor cells.Blood. 2010; 115: 4569-4579Crossref PubMed Scopus (30) Google Scholar]. Each sample was amplified in triplicate.Table1Primers used in real-time polymerase chain reaction analysesGeneOrientationSequenceGLUT1Forward5′-ATACTCATGACCATCGCGCTAG-3′GLUT1Reverse5′-AAAGAAGGCCACAAAGCCAAAG-3′HIF1AForward5′-GAAAGCGCAAGTCTTCAAAG-3′HIF1AReverse5′-TGGGTAGGAGATGGAGATGC-3′MCT1Forward5′-CATGCCACCACCAGCGAAG-3′MCT1Reverse5′-TGACAAGCAGCCACCAACAATC-3′PDK1Forward5′-CCGCTCTCCATGAAGCAGTT-3′PDK1Reverse5′-TTGCCGCAGAAACATAAATGAG-3′PDK2Forward5′-CCGCTGTCCATGAAGCAGTT-3′PDK2Reverse5′-TGCCTGAGGAAGGTGAAGGA-3′PDK3Forward5′-CAAGCAGATCGAGCGCTACTC-3′PDK3Reverse5′-CGAAGTCCAGGAATTGTTTGATG-3′PDK4Forward5′-CCCGAGAGGTGGAGCATTT-3′PDK4Reverse5′-GCATTTTCTGAACCAAAGTCCAGTA-3′28S rRNAForward5′-TGGGTTTTAAGCAGGAGGTG-3′28S rRNAReverse5′-CCAGCTCACGTTCCCTATTA-3′GLUT=Glucose transporter; HIF=hypoxia-inducible factor; LDH=lactate dehydrogenase; MCT=monocarboxylate transporter; PDH=pyruvate dehydrogenase; PDK=pyruvate dehydrogenase kinase. Open table in a new tab GLUT=Glucose transporter; HIF=hypoxia-inducible factor; LDH=lactate dehydrogenase; MCT=monocarboxylate transporter; PDH=pyruvate dehydrogenase; PDK=pyruvate dehydrogenase kinase. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), native PAGE for LDH isozyme separation, and the LDH staining of gels were performed as previously described [30Yonezawa M Nakagawa M Nakamura S et al.Conserved and unique thermodynamic properties of lactate dehydrogenases in an ectothermic organism, the teleost Microstomus achne, and an endothermic organism, bovine.J Biochem. 2016; 160: 299-308Crossref PubMed Scopus (2) Google Scholar, 31Goto T Sugawara K Nakamura S Kidokoro S Wakui H Nunomura W Enzymatic and thermodynamic profiles of a heterotetramer lactate dehydrogenase isozyme in swine.Biochem Biophys Res Commun. 2016; 479: 860-867Crossref PubMed Scopus (6) Google Scholar]. An immunoblot analysis was performed as previously described [4Ubukawa K Guo YM Takahashi M et al.Enucleation of human erythroblasts involves non-muscle myosin IIB.Blood. 2012; 119: 1036-1044Crossref PubMed Scopus (61) Google Scholar, 5Kobayashi I Ubukawa K Sugawara K et al.Erythroblast enucleation is a dynein-dependent process.Exp Hematol. 2016; 44: 247-256Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar]. In the analysis of HIF-1α expression, cells were treated with 100 µmol/L CoCl2, which is a chemical stability enhancer of HIF-1α [32Zhang YB Wang X Meister EA et al.The effects of CoCl2 on HIF-1α protein under experimental conditions of autoprogressive hypoxia using mouse models.Int J Mol Sci. 2014; 15: 10999-11012Crossref PubMed Scopus (30) Google Scholar], at 37°C for 24hours before harvesting, as described in a previous study [27de Heredia FP Wood IS Trayhurn P Hypoxia stimulates lactate release and modulates monocarboxylate transporter (MCT1, MCT2, and MCT4) expression in human adipocytes.Eur J Physiol. 2010; 459: 509-518Crossref PubMed Scopus (108) Google Scholar]. The primary and secondary antibodies used in the present study are listed in Table2. ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) was used for color development. The intensities of immunoreactive protein bands were quantified using the ChemiDoc XRS imaging system (Bio-Rad, Tokyo, Japan). The relative expression levels of the analyzed proteins were normalized with α-tubulin expression.Table2Antibodies used in immunoblot analysesAntigenaThe amino acid sequences of all antigens were derived from human resources.Host animal/Polyclonal or monoclonalbPoly or mono indicates a polyclonal or monoclonal antibody, respectively.Vendor informationα-tubulinMouse/monoSigma-Aldrich, T9026GLUT1Rabbit/polyMillipore, 07-1401HIF-1αRabbit/polyCell Signaling, 3716LDHARabbit/polyCell Signaling, 3582LDHBRabbit/polyGeneTex, GTX101747MCT1Rabbit/polyMillipore, AB3538PPDHMouse/monoAbcam, ab110330PDH phosphorylated at Ser232Rabbit/polyMillipore, AP1063PDH phosphorylated at Ser293Rabbit/polyMillipore, ABS204PDH phosphorylated at Ser300Rabbit/polyMillipore, ABS194PDK1Rabbit/polySigma-Aldrich, D9320PDK2Rabbit/polyBioworld, BS3913PDK3Mouse/monoGeneTex, GTXab55579PDK4Rabbit/polySigma-Aldrich, K4980Mouse IgGGoat/polyHRP-linked, KPL, 074-1809Rabbit IgGGoat/polyHRP-linked, Cell Signaling, 7074GLUT=glucose transporter; HIF=hypoxia-inducible factor; HRP=horseradish peroxidase; IgG, immunoglobulin G; LDH=lactate dehydrogenase; MCT=monocarboxylate transporter; PDH=pyruvate dehydrogenase; PDK=pyruvate dehydrogenase kinase.a The amino acid sequences of all antigens were derived from human resources.b Poly or mono indicates a polyclonal or monoclonal antibody, respectively. Open table in a new tab GLUT=glucose transporter; HIF=hypoxia-inducible factor; HRP=horseradish peroxidase; IgG, immunoglobulin G; LDH=lactate dehydrogenase; MCT=monocarboxylate transporter; PDH=pyruvate dehydrogenase; PDK=pyruvate dehydrogenase kinase. Statistical analyses were performed using Student's t test
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