1710 MOLECULAR MEDICINE REPORTS 18: 1710-1717, 2018 Hypoxia induces lactate secretion and glycolytic efflux by downregulating mitochondrial pyruvate carrier levels in human umbilical vein endothelial cells DONG WANG1*, QINGJIE WANG2, GAOLIANG YAN1, YONG QIAO1, BOQIAN ZHU1, BO LIU1 and CHENGCHUN TANG1* 1Department of Cardiology, Zhongda Hospital of Southeast University Medical School, Nanjing, Jiangsu 210009; 2Department of Cardiology, Changzhou Hospital of Nanjing Medical University, Changzhou, Jiangsu 213004, P.R. China Received December 16, 2017; Accepted May 15, 2018 DOI: 10.3892/mmr.2018.9079 Abstract. The mitochondrial pyruvate carrier (MPC) matrix. In 2012, two independent groups discovered the long complex, located on the inner mitochondrial membrane, sought-after mammalian mitochondrial pyruvate carrier transports pyruvate to the mitochondrial matrix for oxidative (MPC), which is composed of two paralogous subunits, phosphorylation. Previous studies have shown that the MPC MPC1 and MPC2 (1,2). The location of the MPC complex complex is a key regulator of glycolysis in tumor cells. The at the IMM puts it at the intersection between cytosolic present study evaluated the role of the MPC under hypoxic glycolysis and mitochondrial OXPHOS. Subsequent works conditions in human umbilical vein endothelial cells, which confirmed the critical role of the MPC complex in multiple rely on glycolysis for energy generation. It was indicated that cellular functions, including the metabolism of glucose and hypoxia led to an increase in lactate secretion and a decrease pyruvate, fibrosis, and effects on drug efficacy (3-6). Unlike in MPC1 and MPC2 levels, which were upregulated following other mitochondrial carriers that can function as a monomer, re-oxygenation. In addition, the knockdown of MPC1 or pyruvate transport by the MPC complex requires both MPC1 treatment with the MPC inhibitor UK5099 increased the and MPC2 (1). In addition, the function of the MPC complex levels of glycolytic enzymes, HK2, PFKFB3, and LDHA, is closely related to mitochondrial function, especially promoting glycolysis and lactate secretion. Taken together, MPC1, in mammals. the present data revealed that hypoxia can induce lactate Studies show that MPC dysfunction causes various secretion and glycolytic efflux by downregulating MPC diseases, including lactic acidosis, hyperpyruvatemia, tumors levels. and other severe diseases (7). As the gatekeeper for pyruvate entry into mitochondria, the MPC is thought to be of core Introduction position in cell metabolic programming. MPC dysfunction or expression reduction blocks pyruvate entry into the TCA cycle, Oxidative phosphorylation (OXPHOS) is an essential meta- which leads to a metabolism switch to increase glycolysis. bolic pathway for the generation of energy in cells. In most Depending on their localization, endothelial cells (ECs) are eukaryotes, this process takes place in mitochondria and exposed to various oxygen tensions. Like tumor cells, ECs usually requires the cytosolic substrate pyruvate. Extensive rely on glycolysis (specifically, aerobic glycolysis) for energy effort has been put into identifying the molecular carrier production (8). Glycolysis provides bioenergetic intermedi- responsible for the transport of pyruvate across the inner ates, but generates less ATP. In physiological situations, cells mitochondrial membrane (IMM) and into the mitochondrial change from aerobic oxidation to glycolysis in hypoxic condi- tions. While most pyruvate, the primary product of glycolysis, is converted to lactate by lactate dehydrogenase A (LDHA), a small amount of pyruvate is transferred to mitochondria for OXPHOS. Given that the MPC complex represents a Correspondence to: Professor Chengchun Tang, Department of crucial checkpoint in the regulation of cellular metabolism, Cardiology, Zhongda Hospital of Southeast University Medical understanding how it is regulated could have an enormous School, 87 Dingjiaqiao Road, Nanjing, Jiangsu 210009, P.R. China E-mail: [email protected] impact on the treatment of human diseases. Currently, whether and how MPC expression is altered in response to stressful *Contributed equally conditions such as hypoxia is unclear. Knowledge gained from such research will advance our understanding of the roles and Key words: mitochondrial pyruvate carrier, lactate, hypoxia, regulatory mechanisms of the MPC complex in ECs. glycolysis, endothelial cells For a long time, most of the studies on human vascular ECs are based on human umbilical vein ECs (HUVECs), and all the functions of ECs can be achieved through in vitro WANG et al: MITCHONDRIAL PYRUVATE CARRIER AND LACTATE SECRETION IN ENDOTHELIAL CELLS 1711 culture (9). HUVECs provide a classic model system to study Reverse transcription‑polymerase chain reaction (RT‑PCR). many aspects of endothelial function and disease, such as Total RNA was isolated from cells using standard procedure tumor-associated angiogenesis, cardiovascular-related compli- according to the manufacturer's instructions. The RNA was cations, oxidative stress, hypoxia and inflammation related reverse-transcribed to cDNA with random primers using pathways in endothelia, mode of action and cardiovascular All-In-One RT MasterMix (Applied Biological Materials, protection effects of various compounds. Inc., Richmond, BC, Canada) at 25˚C for 10 min, 42˚C for In this study, we initially examined the MPC expression 15 min, followed by 85˚C for 5 min. PCR was performed levels in several metabolic cell types, including HUVECs, using an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA, human coronary artery ECs (HCAECs), human umbilical vein USA). The reaction mixture consisted of 1 µl template cDNA, smooth muscle cells (HUSMCs), and human embryonic kidney 0.2 µM of each primer and 25 µl 2xPCR Taq MasterMix cells 293. Our data indicate that, while MPC1 and MPC2 were (Applied Biological Materials, Inc.). PCR was performed for expressed at significantly higher levels in 293T cells, no signif- 32 cycles for each gene with denaturation at 94˚C for 30 sec, icant differences were observed among HUVECs, HCAECs, annealing at 61˚C for 30 sec, and extension at 72˚C for 20 sec. and HUSMCs. Furthermore, hypoxia was found to increase PCR products were quantified using NIH Image. The primer lactate secretion while it led to reduced MPC1 and MPC2 sequences purchased from Invitrogen for MPC1 (sense: levels in HUVECs. Following re-oxygenation, the levels of 5'-GCC TAC AAG GTA CAG CCT CG-3'; antisense: 5'-GTG both subunits rose. To explore the role of the MPC complex TTT GAT AAG CCG CCC TC-3') and for MPC2 (Accession in cellular metabolism under hypoxia, a small interfering no. NM_001143674.3) (sense: 5'-TAC CAC CGG CTC CTC RNA (siRNA) targeting the mpc1 gene and the MPC inhibitor GAT AA-3'; antisense: 5'-ACA GCA GAT TGA GCT GTG UK5099 were utilized to inhibit MPC1 expression and CT-3'). β-actin (sense: 5'-CCC ATC TAT GAG GGT TAC GC-3'; MPC function. Treatment with either the siRNA or UK5099 antisense: 5'-TTT AAT GTC ACG CAC GAT TTC-3') was used promoted aerobic glycolysis and lactate secretion in HUVECs as a reference gene. under hypoxia. These results indicate that hypoxia can induce lactate secretion and glycolytic flux by downregulating MPC Quantitative PCR (qPCR). qPCR was performed in a final levels. volume of 20 µl containing cDNA template, primers and qRCR MasterMix (Applied Biological Materials, Inc.) using the 7300 Materials and methods qPCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) as described in the manufacture's Cell culture. HCAECs were purchased from Promocell manual. PCR amplification was carried out on 95˚C for 30 sec, (Heidelberg, Germany). HUSMCs and 293T were grown 40 cycles at 95˚C for 5 sec and 60˚C for 31 sec using the in Dulbecco's modified Eagle's minimal essential medium following primers: MPC1, MPC2 and β-actin were described (DMEM) supplemented with 10% fetal bovine serum. above; Fis1 (Accession no. NM_016068.2) (sense: 5'-CTT AAA HUVECs and HCAECs were cultured in endothelial growth GTA CGT CCG CGG GT-3'; antisense: 5'-GCC CAC GAG TCC media-2 (Promocell) supplemented with EC growth supple- ATC TTT CT-3'); Opa1 (Accession no. NM_015560.2) (sense: ment (Promocell) at 37˚C in a humidified atmosphere of 95% 5'-TAC CAG CCT CGC AGG AAT TT-3'; antisense: 5'-CTT TTT air and 5% CO2. To determine the effect of hypoxia-normoxia GGC TGT GTA GCC ACC-3'). The relative mRNA amounts transition on MPC expression, cells were incubated under of target genes were normalized to the values of β-actin. The hypoxia (1% O2 and 99% N2) for 24 h, and they were then results were expressed as fold‑changes of Cquantification cycle -ΔΔCq cultured for 24 h under normoxia (95% air and 5% CO2). (Cq) value relative to the controls using the 2 method. Reagents. JC‑1 fluorescent probe was purchased from Beyotime Western blotting. Western blotting was carried as described Institute of Biotechnology (Jiangsu, China). UK5099 was previously. In brief, cells lysate with equal amount of protein purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, (50 µg) were separated by 10% SDS-polyacrylamide gel Germany). UK5099 was dissolved in Dimethyl sulfoxide electrophoresis and then transferred electronically to the (DMSO), and the final concentration of DMSO was less than polyvinylidene difluoride membranes. Membranes were 0.05%. UK5099 was optimized to a final concentration of blocked in 5% non-fat milk powder in TBST for 1 h at room 40 µM to reduce pyruvate transportation