Hypoxia induces lactate secretion and glycolytic efflux
by downregulating mitochondrial pyruvate carrier
levels in human umbilical vein endothelial cells

Abstract. The mitochondrial pyruvate carrier (MPC)
complex, located on the inner mitochondrial membrane,
transports pyruvate to the mitochondrial matrix for oxidative
phosphorylation. Previous studies have shown that the MPC
complex is a key regulator of glycolysis in tumor cells. The
present study evaluated the role of the MPC under hypoxic
conditions in human umbilical vein endothelial cells, which
rely on glycolysis for energy generation. It was indicated that
hypoxia led to an increase in lactate secretion and a decrease
in MPC1 and MPC2 levels, which were upregulated following
re-oxygenation. In addition, the knockdown of MPC1 or
treatment with the MPC inhibitor UK5099 increased the
levels of glycolytic enzymes, HK2, PFKFB3, and LDHA,
promoting glycolysis and lactate secretion. Taken together,
the present data revealed that hypoxia can induce lactate
secretion and glycolytic efflux by downregulating MPC
levels.
Introduction
Oxidative phosphorylation (OXPHOS) is an essential meta￾bolic pathway for the generation of energy in cells. In most
eukaryotes, this process takes place in mitochondria and
usually requires the cytosolic substrate pyruvate. Extensive
effort has been put into identifying the molecular carrier
responsible for the transport of pyruvate across the inner
mitochondrial membrane (IMM) and into the mitochondrial
matrix. In 2012, two independent groups discovered the long
sought-after mammalian mitochondrial pyruvate carrier
(MPC), which is composed of two paralogous subunits,
MPC1 and MPC2 (1,2). The location of the MPC complex
at the IMM puts it at the intersection between cytosolic
glycolysis and mitochondrial OXPHOS. Subsequent works
confirmed the critical role of the MPC complex in multiple
cellular functions, including the metabolism of glucose and
pyruvate, fibrosis, and effects on drug efficacy (3-6). Unlike
other mitochondrial carriers that can function as a monomer,
pyruvate transport by the MPC complex requires both MPC1
and MPC2 (1). In addition, the function of the MPC complex
is closely related to mitochondrial function, especially
MPC1, in mammals.
Studies show that MPC dysfunction causes various
diseases, including lactic acidosis, hyperpyruvatemia, tumors
and other severe diseases (7). As the gatekeeper for pyruvate
entry into mitochondria, the MPC is thought to be of core
position in cell metabolic programming. MPC dysfunction or
expression reduction blocks pyruvate entry into the TCA cycle,
which leads to a metabolism switch to increase glycolysis.
Depending on their localization, endothelial cells (ECs) are
exposed to various oxygen tensions. Like tumor cells, ECs
rely on glycolysis (specifically, aerobic glycolysis) for energy
production (8). Glycolysis provides bioenergetic intermedi￾ates, but generates less ATP. In physiological situations, cells
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
crucial checkpoint in the regulation of cellular metabolism,
understanding how it is regulated could have an enormous
impact on the treatment of human diseases. Currently, whether
and how MPC expression is altered in response to stressful
conditions such as hypoxia is unclear. Knowledge gained from
such research will advance our understanding of the roles and
regulatory mechanisms of the MPC complex in ECs.
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
Hypoxia induces lactate secretion and glycolytic efflux
by downregulating mitochondrial pyruvate carrier
levels in human umbilical vein endothelial cells
1 Department of Cardiology, Zhongda Hospital of Southeast University Medical School, Nanjing, Jiangsu 210009;
2 Department 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
Correspondence to: Professor Chengchun Tang, Department of
Cardiology, Zhongda Hospital of Southeast University Medical
School, 87 Dingjiaqiao Road, Nanjing, Jiangsu 210009, P.R. China
Contributed equally
Key words: mitochondrial pyruvate carrier, lactate, hypoxia,
glycolysis, endothelial cells
WANG et al: MITCHONDRIAL PYRUVATE CARRIER AND LACTATE SECRETION IN ENDOTHELIAL CELLS 1711
culture (9). HUVECs provide a classic model system to study
many aspects of endothelial function and disease, such as
tumor-associated angiogenesis, cardiovascular-related compli￾cations, oxidative stress, hypoxia and inflammation related
pathways in endothelia, mode of action and cardiovascular
protection effects of various compounds.
In this study, we initially examined the MPC expression
levels in several metabolic cell types, including HUVECs,
human coronary artery ECs (HCAECs), human umbilical vein
smooth muscle cells (HUSMCs), and human embryonic kidney
cells 293. Our data indicate that, while MPC1 and MPC2 were
expressed at significantly higher levels in 293T cells, no signif￾icant differences were observed among HUVECs, HCAECs,
and HUSMCs. Furthermore, hypoxia was found to increase
lactate secretion while it led to reduced MPC1 and MPC2
levels in HUVECs. Following re-oxygenation, the levels of
both subunits rose. To explore the role of the MPC complex
in cellular metabolism under hypoxia, a small interfering
RNA (siRNA) targeting the mpc1 gene and the MPC inhibitor
UK5099 were utilized to inhibit MPC1 expression and
MPC function. Treatment with either the siRNA or UK5099
promoted aerobic glycolysis and lactate secretion in HUVECs
under hypoxia. These results indicate that hypoxia can induce
lactate secretion and glycolytic flux by downregulating MPC
levels.
Materials and methods
Cell culture. HCAECs were purchased from Promocell
(Heidelberg, Germany). HUSMCs and 293T were grown
in Dulbecco’s modified Eagle’s minimal essential medium
(DMEM) supplemented with 10% fetal bovine serum.
HUVECs and HCAECs were cultured in endothelial growth
media-2 (Promocell) supplemented with EC growth supple￾ment (Promocell) at 37˚C in a humidified atmosphere of 95%
air and 5% CO2. To determine the effect of hypoxia-normoxia
transition on MPC expression, cells were incubated under
hypoxia (1% O2 and 99% N2) for 24 h, and they were then
cultured for 24 h under normoxia (95% air and 5% CO2).
Reagents. JC‑1 fluorescent probe was purchased from Beyotime
Institute of Biotechnology (Jiangsu, China). UK5099 was
purchased from Sigma-Aldrich (Merck KGaA, Darmstadt,
Germany). UK5099 was dissolved in Dimethyl sulfoxide
(DMSO), and the final concentration of DMSO was less than
0.05%. UK5099 was optimized to a final concentration of
40 µM to reduce pyruvate transportation into mitochondrial
based on a series of UK5099 dose tested in a range of 10 µM
to 100 µM as previously published (10,11).
Silencing experiments. HUVECs were grown in 6-well plates
up to 85% confluence and transfected using Lipofectamine
RNAiMAX (Invitrogen, Merelbeke, Belgium) with 200 pmol
of specific siRNA (GenePharma, Shanghai, China) targeting
MPC1 (Accession no. NM_001270879.1) (sense: 5′-GGC
UUAUCAAACACGAGAUTT-3′; antisense: 5′-AUCUCG
UGUUUGAUAAGCCTT-3′). All star control siRNA (sense:
5′-UUCUUCGAACGUGUCACGUTT-3′; antisense: 5′-ACG
UGACACGUUCGGAGAATT-3′) was used as negative
control. Silencing efficiency was detected by western blotting.
Reverse transcription‑polymerase chain reaction (RT‑PCR).
Total RNA was isolated from cells using standard procedure
according to the manufacturer’s instructions. The RNA was
reverse-transcribed to cDNA with random primers using
All-In-One RT MasterMix (Applied Biological Materials,
Inc., Richmond, BC, Canada) at 25˚C for 10 min, 42˚C for
15 min, followed by 85˚C for 5 min. PCR was performed
using an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA,
USA). The reaction mixture consisted of 1 µl template cDNA,
0.2 µM of each primer and 25 µl 2xPCR Taq MasterMix
(Applied Biological Materials, Inc.). PCR was performed for
32 cycles for each gene with denaturation at 94˚C for 30 sec,
annealing at 61˚C for 30 sec, and extension at 72˚C for 20 sec.
PCR products were quantified using NIH Image. The primer
sequences purchased from Invitrogen for MPC1 (sense:
5′-GCCTACAAGGTACAGCCTCG-3′; antisense: 5′-GTG
TTTGATAAGCCGCCCTC-3′) and for MPC2 (Accession
no. NM_001143674.3) (sense: 5′-TACCACCGGCTCCTC
GATAA-3′; antisense: 5′-ACAGCAGATTGAGCTGTG
CT-3′). β-actin (sense: 5′-CCCATCTATGAGGGTTACGC-3′;
antisense: 5′-TTTAATGTCACGCACGATTTC-3′) was used
as a reference gene.
Quantitative PCR (qPCR). qPCR was performed in a final
volume of 20 µl containing cDNA template, primers and qRCR
MasterMix (Applied Biological Materials, Inc.) using the 7300
qPCR system (Applied Biosystems; Thermo Fisher Scientific,
Inc., Waltham, MA, USA) as described in the manufacture’s
manual. PCR amplification was carried out on 95˚C for 30 sec,
40 cycles at 95˚C for 5 sec and 60˚C for 31 sec using the
following primers: MPC1, MPC2 and β-actin were described
above; Fis1 (Accession no. NM_016068.2) (sense: 5′-CTTAAA
GTACGTCCGCGGGT-3′; antisense: 5′-GCCCACGAGTCC
ATCTTTCT-3′); Opa1 (Accession no. NM_015560.2) (sense:
5′-TACCAGCCTCGCAGGAATTT-3′; antisense: 5′-CTTTTT
GGCTGTGTAGCCACC-3′). The relative mRNA amounts
of target genes were normalized to the values of β-actin. The
results were expressed as fold‑changes of Cquantification cycle
(Cq) value relative to the controls using the 2-ΔΔCq method.
Western blotting. Western blotting was carried as described
previously. In brief, cells lysate with equal amount of protein
(50 µg) were separated by 10% SDS-polyacrylamide gel
electrophoresis and then transferred electronically to the
polyvinylidene difluoride membranes. Membranes were
blocked in 5% non-fat milk powder in TBST for 1 h at room
temperature, and then incubated with targeting antibodies:
MPC1 (ab74871, dilution 1:1,000; Abcam, Cambridge,
UK), anti-MPC2 (ab111380, dilution 1:1,000; Abcam),
hexokinase II (HK2; ab104836, dilution 1:1,000; Abcam),
LDHA (ab101562, dilution 1:1,000; Abcam), 6-phospho￾fructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3;
ab181861, dilution 1:1,000; Abcam), β-actin (3700, dilution
1:1,000; Cell Signaling Technology, Inc., Danvers, MA,
USA) at 4˚C overnight. Finally, the membranes were incu￾bated with secondary antibodies conjugated with horseradish
peroxidase for 1 h at room temperature. Immunoreactive
materials were visualized by using Chemiluminescent
Substrate kit (Pierce; Thermo Fisher Scientific, Inc.). The
membranes were scanned and the sum optical density was
1712 MOLECULAR MEDICINE REPORTS 18: 1710-1717, 2018
quantitatively analyzed by Quantityone software (Bio-Rad
Laboratories, Inc.).
Lactate concentration measurement. HUVECs were washed
with ice cold PBS three times and split with RIPA for
20 min at 4˚C. Lysates was centrifuged and supernatant was
analyzed by Amplite™ Colorimetricn L-Lactate Assay kit
(AAT Bioquest, Inc., Sunnyvale, CA, USA) according to the
manufacture’s instruction. Lactate concentration in each well
was normalized to total protein content by the Brandford
assay.
ATP production measurement. 1×106 cells were harvested in
200 µl PBS and cell lysate was achieved by sonication before
the homogenate was centrifuged at 12,000 x g for 5 min.
The intracellular ATP production was assessed by using an
Enhanced ATP Assay kit (Beyotime Institute of Biotechnology)
and the manufacturers’ instructions were strictly followed.
Luminescence was measured by a luminometer (Fluroskan
Ascent FL; Thermo Fisher Scientific, Inc.). Data were
normalized based on the protein concentration measured by
the Brandford assay.
Measurement of mitochondrial membrane potential (∆ψm).
Cells cultured in 6-well plates after indicated treatments
were incubated with an equal volume of JC-1 staining solu￾tion (5 µg/ml) at 37˚C for 20 min and rinsed twice with
PBS. Mitochondrial membrane potential was monitored by
determining the relative amounts of dual emissions from mito￾chondrial JC-1 monomers or aggregates using an Olympus
fluorescent microscope under 488 nm laser excitation.
Moreover, the fluorescence intensity was detected with a
flow cytometry (BD FACSCalibur; BD Biosciences, Franklin
Lakes, NJ, USA). The wavelengths of excitation and emission
were 514 and 529 nm for detection of monomeric form of
JC-1. 585 and 590 nm were used to detect aggregation of JC-1.
Mitochondrial depolarization is indicated by an increase in the
green/red fluorescence intensity ratio.
Transmission electron microscope. HUVECs were harvested
using trypsin-EDTA. After washing for three tomes with ice
cold PBS, cells were fixed with 4% glutaraldehyde overnight at
4˚C. A specimen was cut into ultra‑thin sections of 60‑68 nm.
A JEM-1010 model transmission electron microscope (Japan
Electron Optics Laboratory Company, Tokyo, Japan) was used
to observe the ultra-microstructures of mitochondrial.
Mitochondrial pyruvate measurement. Mitochondrial pyru￾vate concentration was determined by pyruvate assay kit
(BioVision, California, USA) according to the instructions.
Briefly, 50 µl working reagent and 50 µl test or standard
sample were mixed, and then added in 96-well plate. After
30 min’s incubation at room temperature, the color intensity
of the reaction product at 570 nm was read and recorded with
a Microplate Reader (Infinite® M200; Tecan Ltd.). The results
were normalized to the total cell numbers.
Statistical analysis. Data were presented as means ± standard
deviation. One-way analysis of variance was used for multiple
comparisons by SPSS 19.0 (SPSS, Inc, Chicago, IL, USA).
If there was a significant variation between treated groups,
Tukey’s post hoc test was applied. P<0.05 was considered to
indicate a statistically significant difference.
Results
MPC expression levels in different metabolic cell types. To
evaluate whether MPC expression varies among different cell
types, we examined the MPC levels in HUVECs, HCAECs,
293T cells, and HUSMCs. The former three cell types rely
on glycolysis for energy production while HUSMCs rely on
oxidation (12,13). Using reverse transcription-polymerase
chain reaction (RT-PCR), both mpc1 and mpc2 were shown
to be expressed in HUVECs, HCAECs, HUSMCs, and 293T
cells (Fig. 1A). qPCR demonstrated that, compared to the
other cell types, MPC1 and MPC2 were highly expressed in
293T cells, a typical anaerobic cell type (13). No significant
difference was observed in MPC expression among HUVECs,
HCAECs, and HUSMCs (Fig. 1B).
The effect of hypoxia on lactate secretion and MPC expres‑
sion in HUVECs. Lactate, generated from pyruvate by
LDHA, is the final product of glycolysis. To determine the
effect of hypoxia on this metabolic process, we measured the
lactate levels as well as MPC expression upon exposure to
hypoxia in HUVECs. An L-lactate acid assay revealed that
the extracellular lactate concentration was increased under
hypoxia for 24 h in HUVECs, while the levels of intracel￾lular lactate were not significantly affected (Fig. 2A). This
suggests that lactate efflux was upregulated under hypoxia.
qPCR and Western blotting showed that the expression of
MPC1 and MPC2 was downregulated under conditions of
hypoxia lasting for 24 h and subsequently induced following
re-oxygenation (Fig. 2B and C). Opa1 and Fis1, two key
factors in mediating mitochondrial fusion and fission, were
used as positive controls as they have been shown to be influ￾enced by extracellular oxygen levels (14). As demonstrated
Figure 1. Determination of MPC1 and MPC2 expression. MPC1 and MPC2
mRNA expression in HUVECs, HUSMCs, HCAEC, 293T cells were detected
by (A) RT-PCR and (B) qPCR. Data are expressed as mean ± standard devia￾tion, n=3. **P<0.01 vs. HUVECs, HUSMC and HCAEC. MPC, mitochondrial
pyruvate carrier; HUVEC, human umbilical vein endothelial cell; HUSMC,
human umbilical vein smooth muscle cell; HCAEC, human coronary artery
endothelial cell.
WANG et al: MITCHONDRIAL PYRUVATE CARRIER AND LACTATE SECRETION IN ENDOTHELIAL CELLS 1713
in Fig. 2D, HUVECs exposed to hypoxia exhibited signifi￾cantly increased mRNA levels of Opa1 and Fis1 at 24 h
post-treatment; further, these levels were increased following
re-oxygenation for 24 h.
Role of the MPC complex in hypoxia‑induced lactate secre‑
tion and aerobic glycolysis in HUVECs. To evaluate whether
hypoxia-induced lactate secretion was related to MPC func￾tion, MPC1 expression was silenced using an siRNA and MPC
activity was inhibited using UK5099 (15). Western blotting
revealed that the siRNA targeting MPC1 (siMPC1) reduced
MPC1 levels specifically without affecting MPC2 expression
(Fig. 3A). It was discovered that UK5099 was optimized to a
final concentration of 40 µM to effectively reduce pyruvate
transportation into mitochondria (Fig. 3B). Both MPC1
silencing and treatment with UK5099 in HUVECs increased
the extracellular lactate concentration significantly, indicating
a higher glycolytic efflux, thus produced more lactic acid
(Fig. 3C). In contrast, ATP production in samples treated
with UK5099 or siMPC1 was significantly lower than that in
control cells (Fig. 3D). Next, to determine how the inhibition
of MPC affects glycolysis, the levels of the key glycolytic
enzymes HK2, LDHA, and PFKFB3 were measured using
Western blotting in HUVECs treated with or without siRNA
or UK5099. As shown in Fig. 3E, exposure to hypoxia led to
the upregulation of HK2, LDHA, and PFKFB3, suggesting
that hypoxia promotes glycolysis. Similarly, treatment with
UK5099 or MPC1 silencing resulted in a significant increase in
the protein expression of HK2, LDHA, and PFKFB3 (Fig. 3E).
The effect of MPC inhibition on mitochondrial structure and
the mitochondrial membrane potential (∆ψm) in HUVECs.
During mitochondrial respiratory oxidation, energy generated
by the electrochemical chain reaction is stored within the
IMM. Changes in this energy generation process can impact
the morphology of the organelle. To determine whether mito￾chondrial structure was affected by the inhibition of MPC,
transmission electron microcopy was utilized to examine the
mitochondrial morphology in HUVECs treated with UK5099
or siRNA. Compared with control treatment groups, MPC1
silencing or UK5099 treatment did not lead to significant
alterations in mitochondrial structure (i.e., no mitochondrial
swelling, pyknosis, and ambiguous cristae were observed;
Fig. 4A).
The mitochondrial ∆ψm, an important parameter
reflecting mitochondrial function (16), can be measured
using the fluorescent dye JC‑1. JC‑1 accumulates and aggre￾gates in healthy mitochondria and can be visualized as red
fluorescence. The treatment of HUVECs with carbonyl
cyanide-m-chlorophenylhydrazone (CCCP), an OXPHOS
inhibitor (17), resulted in an increase in green fluorescence,
indicating a reduction in the mitochondrial ∆ψm (Fig. 4B).
Similarly, the depletion of MPC1 using an siRNA or inhibi￾tion of MPC activity by UK5099 decreased the mitochondrial
∆ψm (Fig. 4B). Dissipation of the mitochondrial ∆ψm can
also be represented by the ratio of aggregated to monomeric
JC-1, since the monomeric form of JC-1 re-localizes to the
cytosol following its disassociation in mitochondria. Using
flow cytometry, a significant amount of JC‑1 aggregates was
observed in the mitochondria of control cells (Fig. 4C). In
contrast, cells treated with CCCP had a lower aggregated
to monomeric JC-1 ratio (Fig. 4C). HUVECs treated with
UK5099 or siMPC1 exhibited a reduced mitochondrial
∆ψm (Fig. 4C). Opa1 and Fis1 mediate mitochondrial fusion
and fission, and are important for the maintenance of the
organelle's function. qPCR revealed that the Opa1 and
Figure 2. Hypoxia increased lactate secretion and decreased MPC in HUVECs. (A) Intracellular and extra cellular lactate concentration under hypoxia
intervention for 24 h. Lactate concentration in each well was normalized to total protein content. MPC1, MPC2 mRNA and protein were detected by (B) qPCR
and (C) western blot analysis, respectively. β-actin was used for normalization. (D) Fis1 and Opa1 mRNA were detected by qPCR. The results are expressed as
mean ± standard deviation, n=3. *
P<0.05, **P<0.01. MPC, mitochondrial pyruvate carrier; HUVEC, human umbilical vein endothelial cell.
1714 MOLECULAR MEDICINE REPORTS 18: 1710-1717, 2018
Fis1 mRNA levels were reduced in MPC1-knockdown and
UK5099-treated HUVECs (Fig. 4D), suggesting a role for
MPC in mitochondrial function.
Discussion
A previous study showed that the MPC complex is a key regu￾lator of glycolysis in tumor cells (11). Independent of the EC
subtype, arterial, venous, lymphatic, and microvascular ECs
rely heavily on glycolysis for energy production regardless
of the abundance of oxygen (8). In this study, we found that
hypoxia decreased MPC1 and MPC2 expression in HUVECs.
This was correlated with an upregulation in the levels of
glycolytic enzymes, leading to increased glycolysis and lactate
secretion.
In contrast to other cell types with greater energy needs,
there is a moderate number of mitochondria in ECs. The
mitochondrial volume in these cells is only 2-6% of the total
cellular volume, compared to 32% in cardiomyocytes and
28% in hepatocytes (16,18,19). Interestingly, we found that
HUVECs, HCAECs, and HUSMCs had similar transcript
levels of MPC1 and MPC2, suggesting that mitochondrial
volume may not be the deciding factor for MPC expression.
Lactate is generated through the metabolism of pyruvate
by LDHA as the final step of glycolysis. The effect of hypoxia
on EC metabolism remains poorly understood. Previous
studies have suggested that hypoxia promotes glycolysis by
upregulating the expression of glycolysis-promoting genes,
stabilizing HIF-1α, inhibiting pyruvate dehydrogenase kinase
and prolyl hydroxylase, and inducing lactate secretion (20).
Recently, De Bock et al (8) demonstrated that glycolysis in
ECs is modulated by the enzyme PFKFB3, an activator of
phosphofructokinase 1, which is a rate-limiting enzyme in
glycolysis. Xu et al (12) later reported that PFKFB3 expression
Figure 3. MPC contributed to hypoxia‑induced lactate secretion and aerobic glycolysis in HUVECs. (A) The efficiency of MPC1 siRNA was verified using
western blotting. β-actin was analyzed for a loading control. (B) Mitochondrial pyruvate concentration after the treatment of UK5099. (C) Intracellular and
extra cellular lactate concentration at different interventions. Lactate concentration in each well was normalized to total protein content. (D) ATP concentra￾tion at different interventions. ATP concentration in each well was normalized to total protein content. (E) The protein level of HK2, LDHA and PFKFB3
were detected by western blot analysis. The β-actin serves as a loading control. The results are expressed as mean ± standard deviation, n=3. *
P<0.05, **P<0.01.
MPC, mitochondrial pyruvate carrier; HUVEC, human umbilical vein endothelial cell; HK2, hexokinase II; LDHA, lactate dehydrogenase A; PFKFB3,
6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3.
WANG et al: MITCHONDRIAL PYRUVATE CARRIER AND LACTATE SECRETION IN ENDOTHELIAL CELLS 1715
was increased upon exposure to hypoxia in ECs. Consistent
with these findings, our data show that HUVECs upregulated
lactate secretion by increasing the levels of the glycolytic
enzymes HK2, LDHA, and PFKFB3 under hypoxic condi￾tions.
Hypoxia signaling can induce a shift from other forms
of energy production to glycolysis-dependent means (21). In
ECs, in which glycolysis is the main form of energy genera￾tion, hypoxia may have a more profound impact on other
metabolic pathways, including mitochondrial OXPHOS. Low
oxygen levels regulate glycolysis-promoting genes while
decreasing pyruvate metabolism (22). Given the important
role of the MPC complex in pyruvate metabolism, alterations
in the expression of MPC could have a significant effect on
cells. We found that the expression of MPC1 and MPC2
was decreased when the oxygen level was low, and that their
expression levels recovered following re-oxygenation. This
suggests that the function of the MPC complex is regulated
by oxygen concentrations. The transportation of pyruvate
into mitochondria by the MPC complex is the first step in
mitochondrial OXPHOS; however, how this process may
affect glycolysis is unclear. Li et al (11) reported that blocking
pyruvate transportation into mitochondria using the MPC
blocker UK5099 attenuated mitochondrial OXPHOS and
triggered aerobic glycolysis in esophageal squamous cell
carcinomas. As pyruvate plays a central role in glucose and
lipid metabolism, we speculated that dysregulation of MPC
function may alter the expression of other metabolic genes
apart from those involved in OXPHOS. Indeed, treatment
with UK5099 or MPC1 silencing upregulated the glycolytic
enzymes HK2, LDHA, and PFKFB3 in HUVECs. However,
the mechanism of MPC inhibition-induced glycolytic
enzymes expression is unclear in ECs. During hypoxia,
hypoxia-inducible factor (HIF) signaling regulates multiple
aspects of ECs metabolism. Stabilization of HIF1α results in
a switch from OXPHOS towards glycolysis by binding to a
hypoxia response element (HRE) in the promoter and thus
upregulating glycolysis-promoting genes, such as HK, glyc￾eraldehyde-3-phosphate dehydrogenase, LDHA and pyruvate
dehydrogenase kinase 1 (PDK1) (22). Furthermore, lactate
production from glycolysis flux can further stabilize HIF1α.
In the present study, we found that treatment with UK5099
or MPC1 silencing increased lactate secretion. Therefore, we
consider that MPC inhibition increases glycolytic enzymes
via both directly and indirectly stabilize HIF1α. A future
work will be performed to fully understand the mechanism.
Due to an insufficient oxygen supply, mitochondrial
OXPHOS is inhibited. ATP levels are also decreased during
the initial phase of hypoxia as a result of reduced glycolysis.
As glycolysis recovers, the production of ATP increases as
long as sufficient amounts of substrates are available (23),
while the concentration of lactate rises steadily. Our data
reveal a significant decrease in ATP synthesis and increase
in lactate production in HUVECs treated with UK5099 or
siMPC1. This suggests that impaired mitochondrial pyruvate
transport could drive glycolysis to promote lactate secretion
in HUVECs.
Mitochondrial function is closely related to the amount
of pyruvate entering mitochondria (16). In our study, we
showed that the mitochondrial ∆ψm dropped when MPC1 was
silenced or when MPC function was inhibited, suggesting that
hypoxia-induced mitochondrial dysfunction is related to MPC
Figure 4. MPC inhibition decreased mitochondrial membrane potential (∆ψm) but did not influence mitochondria structure in HUVECs. (A) The mitochondria
morphology was determined at different interventions for 24 h by FEM. (B) The mitochondrial membrane potential was determined by JC-1 staining. Red
fluorescence represents the mitochondrial aggregate form of JC‑1, indicating intact mitochondrial membrane potential. Green fluorescence represents the
monomeric form of JC-1, indicating dissipation of mitochondrial membrane potential. CCCP was the positive control. Scale bar is 50 µM. (C) The ratio
of red (PI) to green (FITC) fluorescence were checked by flow cytometry. (D) Fis1 and Opa1 mRNA were detected by qPCR. The results are expressed as
mean ± standard deviation, n=3. *
P<0.05, **P<0.01. MPC, mitochondrial pyruvate carrier; HUVEC, human umbilical vein endothelial cell; CCCP, carbonyl
cyanide‑m‑chlorophenylhydrazone; PI, propidium iodide; FITC, fluorescein isothiocyanate.
1716 MOLECULAR MEDICINE REPORTS 18: 1710-1717, 2018
expression and/or activity. Interestingly, little change was
detected in mitochondrial morphology. However, we cannot
rule out the possibility that the inhibition of MPC could induce
changes in organelle structure if treatment was prolonged (>
24 h).
Fission and fusion mechanisms regulate mitochondrial
morphology and apoptosis(14). Fission frequency is determined
by the levels of Fis1, which localizes to the outer mitochondrial
membrane. Fusion frequency is influenced by Opa1 levels,
which are known to be associated with the IMM (24). It has
been shown that the depletion of Opa1 leads to a reduction in
mitochondrial ∆ψm, while fragmentation of the mitochondrial
network by Fis1 leads to cytochrome c release (14). Other
studies have indicated that hypoxia/re-oxygenation results
in a significant reduction in Opa1 levels and upregulation of
Fis1 in cardiomyocytes (25) and hippocampal neurons (26).
However, Chitra and Boopathy (27) reported that hypobaric
hypoxia modulated mitochondrial dynamics by decreasing
Fis1 in rat lung cells. Our data demonstrate that Opa1 and
Fis1 mRNA levels were reduced after MPC1 knockdown or
UK5099 treatment. Additional experimental investigations are
needed to better understand the role of the MPC complex in
mitochondrial fission and fusion.
In conclusion, we found that the MPC complex may play an
essential role in hypoxia-induced glycolysis and lactate secre￾tion in HUVECs. The depletion of MPC1 or inhibition of the
MPC complex leads to increased lactate production, potentially
by upregulating glycolytic enzymes and therefore promoting
glycolysis. This study is the first attempt to link hypoxia to
the MPC complex. It reveals MPC as a potential target for the
treatment of hypoxia-related injury to ECs, including acute
myocardial infarction and pulmonary hypertension. Limiting
glycolysis decreased endothelial sprouting (28), showing the
role of glycolysis on angiogenesis. As we known, angiogen￾esis has close relationship with cancer. It is thus tempting to
speculate that MPC may be a novel target for the prevention
and treatment of cancer. In conclusion, we demonstrated that
hypoxia can induce lactate secretion and glycolytic efflux
by downregulating MPC UK 5099 levels. Our study provided the
evidence that MPC complex may play an essential role in
hypoxia-induced glycolysis and lactate secretion in HUVECs.
MPC might be a novel treatment target for hypoxia-induced
EC injury, such as acute myocardial infarction and pulmonary
hypertension.
Acknowledgements
Funding
The present work was supported by Chengchun Tang
(NSF nos. 81670237 and 81370225) and Dong Wang (the
Fundamental Research Funds for the Central Universities,
no. 2242018K40159).
Availability of data and materials
The datasets used during the present study are available from
the corresponding author on reasonable request.
Authors’ Contributions
DW, QW and CT designed the research. DW, QW, BZ and BL
performed the experiments. DW, QW, GY and YQ analyzed the
data. DW and CT wrote the manuscript. All authors reviewed
and edited the manuscript..
Competing interests
The authors declare that they have no competing interests.
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