Homocysteine Activates B Cells via
Regulating PKM2-Dependent Metabolic
Reprogramming
This information is current as
of June 18, 2017.
Jiacheng Deng, Silin Lü, Huiying Liu, Bo Liu, Changtao
Jiang, Qingbo Xu, Juan Feng and Xian Wang
J Immunol 2017; 198:170-183; Prepublished online 30
November 2016;
doi: 10.4049/jimmunol.1600613
http://www.jimmunol.org/content/198/1/170
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Supplementary
Material
The Journal of Immunology
Homocysteine Activates B Cells via Regulating
PKM2-Dependent Metabolic Reprogramming
Jiacheng Deng,* Silin L€
u,* Huiying Liu,* Bo Liu,* Changtao Jiang,* Qingbo Xu,†
Juan Feng,* and Xian Wang*
H
omocysteine (Hcy) is a sulfur-containing amino acid
formed during the metabolism of the essential amino
acid methionine. Accumulating evidence suggests that
hyperhomocysteinemia (HHcy) is an independent risk factor for
cardiovascular diseases in which inflammation plays a key role (1,
2). Our previous studies have shown that HHcy accelerates early
atherosclerotic lesion formation in apolipoprotein E–deficient
(ApoE2/2) mice and that Hcy stimulation in vitro and ex vivo can
induce B cell proliferation and IgG Ab secretion (3–5). However,
the direct effects of HHcy on B cell function in vivo, the underlying
*Department of Physiology and Pathophysiology, School of Basic Medical Sciences,
Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, People’s Republic of China;
and †Cardiovascular Division, British Heart Foundation Centre for Vascular Regeneration, King’s College London, London SE5 9NU, United Kingdom
Received for publication April 7, 2016. Accepted for publication October 24, 2016.
This work was supported by National Natural Science Foundation of China Grants
91439206 and 31230035 (to X.W.), and 81370006 (to J.F.), and by Chinese Ministry
of Education (111 Project) Grant B07001.
Address correspondence and reprint requests to Prof. Xian Wang and Dr. Juan Feng,
Department of Physiology and Pathophysiology, School of Basic Medical Sciences,
Peking University Health Science Center, 38 Xueyuan Road, Haidian District, Beijing
100191, People’s Republic of China. E-mail addresses: [email protected] (X.W.)
and [email protected] (J.F.)
The online version of this article contains supplemental material.
Abbreviations used in this article: ApoE2/2 , apolipoprotein E–deficient; 2-DG,
2-deoxy-D-glucose; ECAR, extracellular acidification rate; Hcy, homocysteine; HHcy,
hyperhomocysteinemia; IRF4, IFN regulatory factor 4; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LDH, lactate dehydrogenase; 2-NBDG, 2-(N(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose; OCR, oxygen consumption
rate; 3PG, 3-phosphoglycerate; PKM1, pyruvate kinase muscle isozyme 1; PKM2,
pyruvate kinase muscle isozyme 2; siRNA, small interfering RNA; SKN, shikonin;
SRC, spare respiratory capacity; S6RP, S6 ribosomal protein.
This article is distributed under The American Association of Immunologists, Inc.,
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Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600613
mechanisms, and the potential pathophysiological significance
remain to be elucidated.
Recent studies have revealed the interaction of multiple pathways in the regulation of immune and metabolic systems (6).
Alterations in metabolism at both the cellular and tissue level
affect specific lymphocyte functions (6). The Warburg effect, or
aerobic glycolysis, was first discovered in highly proliferating
tumor cells (7). Recently, similar metabolic changes have also been
observed in immune cells. Activated dendritic cells, M1 macrophages, and effector T cells can switch their metabolic program
from oxidative phosphorylation to aerobic glycolysis to meet the
bioenergetic and biosynthetic demands of cell growth or effector
functions (6, 8, 9). Although B cells share several features with
T cells, it has recently been reported that B cells increase their rate
of both glycolysis and oxidative phosphorylation in a relatively
balanced fashion upon BCR or LPS stimulation (10). Moreover, in
the intestinal immune system, IgA+ plasma cells in the intestinal
lamina propria use both glycolytic and oxidative metabolism,
whereas naive B cells in Peyer’s patches preferentially use oxidative metabolism (11). These investigations have revealed an
important role of metabolic reprogramming in B cell activation.
Glucose metabolism is important for B cell activation (12). Pyruvate kinase is one of the key enzymes in the glycolytic pathway.
There are four mammalian pyruvate kinase isoforms. Pyruvate kinase muscle isozyme 2 (PKM2) is mainly expressed in embryonic
cells and tumor cells, whereas pyruvate kinase muscle isozyme
1 (PKM1) is found in highly differentiated tissues, such as muscles
and the brain. The pyruvate kinase RBC isozyme and pyruvate
kinase liver isozyme are tissue-specific isoforms and are found in
RBCs (pyruvate kinase RBC isozyme) or in liver and kidney cells
(pyruvate kinase liver isozyme) (13). Of all these isoforms, PKM2
has been the most extensively studied in tumor cells and has been
found to be critical for tumor cell growth (14–16). The expression
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The overactivation of immune cells plays an important role in the pathogenesis of hyperhomocysteinemia (HHcy)-accelerated
atherosclerosis. Homocysteine (Hcy) activates B cell proliferation and Ab secretion; however, the underlying mechanisms for these
effects remain largely unknown. Metabolic reprogramming is critical for lymphocyte activation and effector function. In this
study, we showed that Hcy-activated B cells displayed an increase in both oxidative phosphorylation and glycolysis, with a
tendency to shift toward the latter, as well as an accumulation of intermediates in the pentose phosphate pathway, to provide
energy and biosynthetic substrates for cell growth and function. Mechanistically, Hcy increased both the protein expression and
glycolytic enzyme activity of the pyruvate kinase muscle isozyme 2 (PKM2) in B cells, whereas the PKM2 inhibitor shikonin
restored Hcy-induced metabolic changes, as well as B cell proliferation and Ab secretion both in vivo and in vitro, indicating
that PKM2 plays a critical role in metabolic reprogramming in Hcy-activated B cells. Further investigation revealed that the
Akt–mechanistic target of rapamycin signaling pathway was involved in this process, as the mechanistic target of rapamycin
inhibitor rapamycin inhibited Hcy-induced changes in PKM2 enzyme activity and B cell activation. Notably, shikonin treatment effectively attenuated HHcy-accelerated atherosclerotic lesion formation in apolipoprotein E–deficient mice. In conclusion, our results demonstrate that PKM2 is required to support metabolic reprogramming for Hcy-induced B cell activation
and function, and it might serve as a critical regulator in HHcy-accelerated initiation of atherosclerosis. The Journal of
Immunology, 2017, 198: 170–183.
The Journal of Immunology
Materials and Methods
Mice and animal models
Six-week-old C57BL/6J mice and ApoE2/2 mice were purchased from the
Animal Center of Peking University Health Science Center (Beijing,
China) and were maintained under specific pathogen-free conditions. For
the induction of HHcy in mice, C57BL/6J mice were fed a normal mouse
chow diet and were provided drinking water supplemented with or without
1.8 g/l DL-Hcy (Sigma-Aldrich, St. Louis, MO) for 2 wk as previously
described (5). In the treatment study, ApoE2/2 mice were i.p. injected with
1.2 mg/kg shikonin (SKN; Sigma-Aldrich) or solvent control every 3 d.
Three days after the first injection, ApoE2/2 mice were fed a normal
mouse chow diet and provided water supplemented with or without 1.8 g/l
Hcy for 2 wk. All studies were performed according to protocols approved
by the Committee on the Ethics of Animal Experiments of Peking University Health Science Center. The investigation conformed to the Guide
for the Care and Use of Laboratory Animals published by the National
Institutes of Health.
Cell isolation and culture
Splenic B cells were isolated from mice and purified via positive selection
using magnetic microbeads against CD19 (Miltenyi Biotec, Bergisch
Gladbach, Germany) according to the manufacturer’s protocol. Purified
B cells were cultured in RPMI 1640 medium (Life Technologies,
Gaithersburg, MD) supplemented with 10% FBS (Gemini Bio-Products, West
Sacramento, CA) and maintained with 0.1 mg/ml LPS (Sigma-Aldrich).
B cells were incubated with or without 100 mM Hcy or 5 mg/ml LPS for
the indicated times. For some experiments, B cells were pretreated with
0.5 mM 2-deoxy-D-glucose (2-DG; Sigma-Aldrich), 0.25 mM SKN, or 10 nM
rapamycin (Sigma-Aldrich) for 30 min before Hcy stimulation.
Transfection of small interfering RNA
Scramble small interfering RNA (siRNA) and siRNA targeting Pkm2 (59CTTGCAGCTATTCGAGGAA-39) were designed by and purchased from
RiboBio (Guangzhou, China). Scramble and PKM2 siRNA were transfected into B cells using an Amaxa mouse B cell Nucleofector kit (Lonza
Cologne, Cologne, Germany) according to a modified version of the
manufacturer’s protocol.
Western blot analysis
Cell extracts containing equal amounts of total protein were resolved via
10% SDS-PAGE and were then transferred to a nitrocellulose membrane.
After blocking with 5% BSA for 1 h, the membrane was incubated with
different primary Abs at 4˚C overnight, including anti-PKM2, anti-PKM1,
anti–IFN regulatory factor 4 (IRF4), anti–phospho-mechanistic target of
rapamycin (mTOR; Ser2448), anti-mTOR, anti–phospho-AKT (Ser473),
anti-AKT, anti–phospho-S6 ribosomal protein (S6RP; Ser235/236), anti–bactin, anti-GAPDH (1:1000; all from Cell Signaling Technology, Danvers,
MA), and anti–eukaryotic translation initiation factor 5 (1:1000; Santa
Cruz Biotechnology, Santa Cruz, CA). The membrane was then incubated
with IRDye 700– or IRDye 800–conjugated secondary Abs (1:20,000;
Rockland, Gilbertsville, PA) for 1 h at room temperature. The fluorescence
signal was detected and analyzed using an Odyssey infrared imaging
system (LI-COR Biosciences, Lincoln, NE).
Quantitative RT-PCR and analysis of Pkm splicing
Total RNA was extracted using TRIzol reagent (Life Technologies, Grand
Island, NY) and was then reverse transcribed to cDNA and amplified using
the AMV reverse transcription system (Promega, Madison, WI). For
quantitative PCR, all amplification reactions involved the use of the
Mx3000 multiplex quantitative PCR system (Stratagene, La Jolla, CA) and
SYBR Green I reagent with results normalized to b-actin mRNA levels.
Detection of postswitch transcripts was performed as described in a previous study (21). The primer sequences used for PCR analyses based on
mouse genes are as follows: Aicda (forward, 59-AAGGGACGGCATGAGACCTA-39; reverse, 59-GCCGAAGTTGTCTGGTTAGC-39), Im-Cg3
(forward, 59-CTCTGGCCCTGCTTATTGTTG-39; reverse, 59-CTCAGGGAAGTAGCCTTTGACA-39), Im-Cg1 (forward, 59-CTCTGGCCCTGCTTATTGTTG-39; reverse, 59-GGATCCAGAGTTCCAGGTCACT-39),
Im-Cg2b (forward, 59-CTCTGGCCCTGCTTATTGTTG-39; reverse, 59CACTGAGCTGCTCATAGTGTAGAGTC-39), Pkm (forward, 59-TCGAGAACCATGAAGGCGTC-39; reverse, 59-ACTTGGTGAGCACTCCTG39), Pkm2 (forward, 59-TCGAGAACCATGAAGGCGTC-39; reverse, 59CGGCGGAGTTCCTCGAATAG-39), Icam-1 (forward, 59-AGCTCGGAGGATCACAAA-39; reverse, 59-TCTGCTGAGACCCCTCTTG-39),
Vcam-1 (forward, 59-CTGTTCCAGCGAGGGTCTA-39; reverse, 59CACAGCCAATAGCAGCACA-39), Tnf-a (forward, 59-ACAGAAAGCATGATCCGCGAC-39; reverse, 59-CCGATCACCCCGAAGTTCAGTA39), Ifn-g (forward, 59-TGGCTGTTTCTGGCTGTTAC-39; reverse, 59TTCGCCTTGCTGTTGCTGAAG-39), Mcp-1 (forward, 59-CAGATGCAGTTAACGCCC-39; reverse, 59-ATTCCTTCTTGGGGTCAGC-39), Il-2
(forward, 59-CAGGAACCTGAAACTCCCCA-39; reverse, 59-AGAAAGTCCACCACAGTTGC-39), b-actin (forward, 59-GTGACGTTGACATCCGTAAAGA-39; reverse, 59-GCCGGACTCATCGTACTCC-39). The Pkm
splicing experiment was performed based on modified methods from a
previous report (22). PCR products were digested with or without NcoI and
PstI (both from Takara Bio, Shiga, Japan) to distinguish Pkm1 and Pkm2
isoforms. The products were then resolved on an agarose gel and analyzed
using the GeneGenius bio-imaging system (Syngene, Nuffield Road, Cambridge, U.K.).
ELISA
For the analysis of cell culture supernatants, B cells (1 3 106 cells per well)
were seeded in a 48-well plate for the indicated times and supernatants
from the cell cultures were harvested. The IgM and IgG levels of the
culture supernatants (1:10 for IgM and 1:1 for IgG) or blood plasma
(1:5,000 for IgM and 1:50,000 for IgG) were analyzed and quantified using
mouse-specific IgM and IgG ELISA kits (Bethyl Laboratories, Montgomery, TX) according to the manufacturer’s protocol.
Extracellular flux analysis
Oxygen consumption rate (OCR) and extracellular acidification rate
(ECAR) were analyzed using an XF24 extracellular flux analyzer (Seahorse
Bioscience, Billerica, MA). B cells (1 3 106 cells per well) were seeded in
an XF24 microplate coated with polylysine (Sigma-Aldrich). Mitochondrial stress tests were performed under basal conditions and in response to
metabolic reagents, including 1 mM oligomycin, 1 mM carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP), 1 mM rotenone, and 1 mM
antimycin A (all from Sigma-Aldrich). Glycolysis stress tests were performed under basal conditions and in response to 10 mM glucose, 1 mM
oligomycin, and 100 mM 2-DG (all from Sigma-Aldrich). Wave software
and the XF Mito/Glycolysis stress test report generator (Seahorse Bioscience) were used to analyze the OCR in mitochondrial stress tests and
the ECAR in glycolysis stress tests. Additionally, for the analysis of ECAR
in mitochondrial stress tests, basal and maximal ECARs were calculated as
previously described (23). Briefly, basal ECAR was the basal rate before
the addition of metabolic reagents, and maximal ECAR was assessed after
the addition of rotenone and antimycin A. Glycolytic reserve was calculated by subtracting basal ECAR from maximal ECAR.
Analysis of metabolites via liquid chromatography–tandem
mass spectrometry
For metabolite extraction, cultured cells were washed with PBS twice and
lysed in 80% aqueous methanol (v/v) equilibrated at 280˚C. [5-13C]glutamine (Cambridge Isotope Laboratories, Tewksbury, MA) was added as
an internal standard. Cell supernatants of metabolite extracts were collected, dried, and stored at 280˚C until used for metabolomic analyses.
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of PKM2 in tumor cells allows for an increase in both glycolytic
and anabolic metabolic rates to support cell growth and proliferation (14). There have been a few recent reports showing that PKM2
is also required for normal cells (17–20). M1 macrophages upregulate PKM2 expression to increase glycolytic flux in support of cell
activation (18, 19). Upon activation, B cells increase their cellular
metabolism and proliferate rapidly. However, whether cellular
metabolism is changed during HHcy-induced B cell activation is
unclear, and if it is changed, the underlying mechanism is unknown.
In this study, we demonstrate that HHcy induces B cell proliferation and Ab secretion both in vivo and in vitro. PKM2 expression and enzyme activity were increased in HHcy-induced
B cells to promote metabolic reprogramming, with an increase
in both oxidative phosphorylation and glycolysis. The inhibition of
PKM2 effectively reversed HHcy-induced B cell proliferation, Ab
secretion, and the early stage of atherogenesis in ApoE2/2 mice.
Therefore, our results suggest that PKM2 is a critical metabolic
regulator of HHcy-induced B cell activation and may serve as a
potential therapeutic target in treating HHcy-related atherosclerosis and B cell–associated inflammatory diseases.
171
172
METABOLIC REPROGRAMMING IN Hcy-INDUCED B CELL ACTIVATION
For liquid chromatography–tandem mass spectrometry (LC-MS/MS),
samples were reconstituted in water and analyzed using a QTRAP 6500
LC-MS/MS System (AB SCIEX, Concord, ON, Canada). MultiQuant v3.0
software (AB SCIEX) was used to process all raw liquid chromatography–
mass spectrometry data and integrate chromatographic peaks. Integrated
peak areas corresponding to metabolite concentrations were further analyzed
using MetaboAnalyst software (24). Metabolite abundance was expressed
relative to the internal standard and was normalized to the number of cells.
Measurement of pyruvate kinase activity
Pyruvate kinase activity was measured using a lactate dehydrogenase
(LDH)-linked assay as previously described (25). B cells were lysed in a
pyruvate kinase lysis buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA,
150 mM NaCl, 1% IGEPAL CA-630) supplemented with protease inhibitors and PMSF. Protein concentrations of the cell lysates were determined,
and 2 mg of total protein was mixed with the reaction mixture (50 mM
Tris-HCl [pH 7.5], 100 mM KCl, 5 mM MgCl2, 0.6 mM ADP, 0.5 mM
phosphoenolpyruvate, 450 mM NADH, and 8 U LDH). The final reaction
volume was 200 ml, and reactions were performed in 96-well plates. The
decrease in absorbance by NADH at 340 nm was monitored and measured
to determine pyruvate kinase activity using a Varioskan flash multimode
reader (Thermo Fisher Scientific, Waltham, MA).
To assess glucose uptake, B cells were cultured and treated as indicated, and
2-NBDG (Life Technologies) was then added into the cell culture medium,
and cultures were incubated for 1 h. For CD138 staining, cells were collected and stained with a PE-conjugated CD138 Ab (BD Pharmingen, San
Diego, CA). Plasma inflammatory cytokine levels were measured using a
cytometric bead array mouse inflammation kit (BD Biosciences, San Jose,
CA). For cell proliferation, B cells were stained with CFSE (Molecular
Probes, Eugene, OR) prior to culture and analyzed at certain times. Flow
cytometry was performed using a FACSCalibur flow cytometer (BD Biosciences) and the data were analyzed using FlowJo software (Tree Star,
Ashland, OR). Cells labeled with 2-NBDG were also analyzed using a
FlowSight imaging flow cytometer (Amnis, Seattle, WA). Bright-field and
fluorescent images were acquired and data analyses were completed using
IDEAS software (Amnis).
Measurement of plasma Hcy and lipid profiles
Plasma Hcy levels were quantified via an enzymatic cycling assay as
previously described (26). Total plasma triglycerides and cholesterol levels
were analyzed using kits from BioSino Biotechnology & Science (Beijing,
China) according to the manufacturer’s protocols.
Analysis of atherosclerotic lesions
Hearts were collected and prepared as previously described (26). Briefly,
hearts were collected and embedded in OCT compound (Tissue-Tek;
Sakura Finetek, Torrance, CA) and frozen. Aortic roots were serially
cross-sectioned at 7-mm intervals, collected on glass slides, and then
stained with Oil Red O to visualize lesion areas.
Statistical analysis
All data are presented as the mean 6 SEM unless otherwise stated.
Comparisons of data sets were performed using unpaired Student t tests for
comparing two groups and one-way ANOVAs followed by Newman–Keuls
post hoc tests for comparing multiple groups. Statistical analyses were
performed using GraphPad Prism (GraphPad Software, La Jolla, CA). A
p value ,0.05 was considered statistically significant for all experiments.
Results
HHcy induces B cell proliferation and Ab secretion both in vivo
and in vitro
We have previously reported that B cells from ApoE2/2 mice with
HHcy show an increase in both cell proliferation and IgG secretion in response to LPS stimulation ex vivo compared with those
from control ApoE2/2 mice (3, 4). To investigate whether HHcy
could regulate normal B cell function in vivo, we fed C57BL/6J
mice a normal chow diet and provided them drinking water supplemented with or without 1.8 g/l Hcy for 2 wk. The results show
that mice given Hcy-supplemented water developed moderate HHcy
compared with control mice (33.58 6 1.28 versus 8.14 6 0.42 mM;
Metabolic reprogramming in B cells stimulated by HHcy
in vivo and in vitro
Upon activation, lymphocytes proliferate, differentiate, and exert
effector functions. Activated lymphocytes need to alter their
metabolic program to produce enough energy and biosynthetic
substrates to meet the demands of cell growth and function (29). To
identify whether metabolic reprogramming is required in HHcyactivated B cells, we first measured the metabolic parameters of
B cells purified from control and HHcy mice using an extracellular
flux analyzer. Analysis revealed that OCR and ECAR were significantly increased in HHcy-treated B cells (Fig. 2A, 2B, upper
panels). The increase in basal OCR (from 124.2 6 5.4 pmol/min
in control cells to 181.2 6 11.7 pmol/min in HHcy-treated cells)
and ECAR (from 3.1 6 0.4 mpH/min in control cells to 6.1 6
0.3 mpH/min in HHcy-treated cells) revealed the activation of
both glycolytic and oxidative metabolism in HHcy-stimulated
B cells, whereas the increases in maximal OCR (from 208.2 6
3.4 pmol/min in control cells to 253.4 6 26.9 pmol/min in HHcytreated cells) and ECAR (from 5.5 6 0.4 mpH/min in control cells
to 10.0 6 0.5 mpH/min in HHcy-treated cells) reflected a higher
maximal metabolic potential in HHcy-treated B cells (Fig. 2A, 2B,
lower panels). Spare respiratory capacity (SRC) and glycolytic
reserve are measurements that reflect the metabolic capacity in
oxidative and glycolytic metabolism, respectively. HHcy-treated
cells had similar spare respiratory capacity but significant higher
glycolytic reserve (Fig. 2A, 2B, lower panels), demonstrating an
elevated metabolic capacity in glycolytic metabolism rather than
oxidative metabolism. In comparing the ratio of OCR to ECAR,
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Flow cytometry
p , 0.05). In the treated mice, HHcy significantly increased B cell
numbers in the spleen (Fig. 1A). Both plasma IgM (from 0.22 6 0.01
to 0.41 6 0.06 mg/ml) and IgG (from 0.95 6 0.04 to 2.21 6
0.40 mg/ml) levels were upregulated in HHcy mice (Fig. 1B, 1C).
These data indicate that HHcy activates B cells in vivo.
The terminal differentiation of B cells into Ab-secreting plasma
cells depends on the expression of several transcription factors (27),
whereas the activation of Ig class switch recombination allows for
genetic alterations of Ig genes and the generation of different Ab
classes (28). We further tested whether Hcy regulated these
processes during B cell activation. Protein expression of IRF4,
which is the key regulator of B cell differentiation, was increased
by ∼70% in HHcy-treated B cells (Fig. 1D). Quantitative PCR
analysis also showed that HHcy-treated B cells had a significantly
elevated mRNA expression of Aicda (Fig. 1E, left), which is
critical for the initiation of class switch recombination (21).
Moreover, the expression of postswitch transcripts, including
Im-Cg2b and Im-Cg1, was increased in HHcy-treated B cells,
whereas Im-Cg3 had a slight tendency for increased expression
(Fig. 1E, right). Thus, HHcy activated B cells through the regulation of terminal differentiation-related transcription factors
and class switch recombination in vivo.
Our in vitro studies further confirmed the results of the in vivo
experiments. Consistent with our previous findings, B cells proliferated at 48 h (Fig. 1F) and secreted IgM and IgG Abs at 72 h
(Fig. 1G, 1H) when treated with Hcy (100 mM). Intriguingly, Hcy
markedly enhanced B cell differentiation into CD138+ cells (from
2.37 6 0.09% in control cells to 6.94 6 0.39% in Hcy treatment
cells; Fig. 1I), which was accompanied by increased protein expression of IRF4 (Fig. 1J). Additionally, Hcy significantly upregulated the mRNA levels of Aicda, Im-Cg2b, and Im-Cg3 in
B cells, whereas Im-Cg1 showed a tendency for increased expression (Fig. 1K). Collectively, these results show that HHcy can
induce B cell proliferation and Ab secretion both in vivo and
in vitro.
The Journal of Immunology
173
an indicator for metabolic switching, we found that HHcyactivated B cells depended more on glycolytic metabolism than
on oxidative metabolism, as evidenced by a 35% reduction in the
OCR/ECAR ratio in HHcy-treated cells compared with that of the
control cells (Fig. 2C, lower panel). These results indicate dramatic metabolic reprogramming in HHcy-treated B cells.
Similar results were obtained when we performed in vitro experiments. Both OCR (141.6 6 0.4 versus 91.4 6 8.0 pmol/min
for basal OCR, 274.7 6 6.5 versus 139.7 6 16.1 pmol/min for
maximal OCR) and ECAR (2.8 6 0.1 versus 1.0 6 0.4 mpH/min
for glycolysis, 3.6 6 0.2 versus 0.9 6 0.3 mpH/min for glycolytic
capacity) were significantly increased in B cells treated with Hcy
(100 mM) for 24 h relative to the control cells (Fig. 2D, 2E). Hcystimulated B cells showed higher metabolic capacity in oxidative
and glycolytic metabolism, with both increased SRC and glycolytic reserve (Fig. 2D, 2E). The OCR/ECAR ratio tended to be
lower in Hcy-treated B cells, although the difference was not
statistically significant (29.6 6 3.9 versus 41.4 6 7.8; Fig. 2F,
lower panel). As glucose is a major source for both glycolytic and
oxidative metabolism, we next assessed glucose uptake using a
fluorescent glucose analog (2-NBDG). Hcy increased glucose
uptake in B cells, as evidenced by fluorescent images and quantification of fluorescence intensity from flow cytometry analysis
(Fig. 2G, 2H). Taken together, these results suggest that both
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FIGURE 1. HHcy induces B cell proliferation and Ab secretion both in vivo and in vitro. (A–E) C57BL/6J mice were fed a normal chow diet and given
drinking water supplemented with or without 1.8 g/l Hcy for 2 wk. (A) Total cell numbers of splenic B cells purified from control or HHcy mice were
counted. Plasma IgM (B) and IgG (C) levels were measured via ELISA. (D) IRF4 protein expression and quantification were analyzed via Western blot.
b-Actin served as an internal control. (E) Gene expression of Aicda and postswitch transcripts (Im-Cg3, Im-Cg2b, Im-Cg1) were measured via quantitative
PCR in B cells. (F–K) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or without 100 mM Hcy for the indicated times. (F) Purified
B cells were labeled with CFSE prior to culture and cell proliferation was assessed by flow cytometry after 48 h. IgM (G) and IgG (H) levels in the culture
supernatants after Hcy stimulation for 72 h were measured via ELISA. (I) After Hcy stimulation for 48 h, cells were labeled with CD138 and then analyzed
via flow cytometry. (J) IRF4 protein expression and quantification in B cells at 24 h were analyzed via Western blot. (K) Gene expression of Aicda and
postswitch transcripts were measured via quantitative PCR in B cells at 72 h. The data shown are representative [(A–D) and (F–I); upper panel in (J)] and
cumulative [(E and K); lower panel in (J)] of at least three independent experiments [n = 3–5 mice in each group in (A–E)]. The data are presented as the
mean 6 SEM. *p , 0.05 compared with the control.
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METABOLIC REPROGRAMMING IN Hcy-INDUCED B CELL ACTIVATION
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FIGURE 2. Metabolic reprogramming in B cells induced by HHcy in vivo and in vitro. (A–C) Metabolic parameters of splenic B cells purified from control or
HHcy C57BL/6J mice were analyzed using an extracellular flux analyzer. (D–H) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or without
100 mM Hcy for 24 h. OCR and ECAR in B cells were determined via extracellular flux analysis. (A and D) The OCR over time was measured at a basal level and
after the injection of oligomycin, FCCP, antimycin A, and rotenone. Basal OCR was determined before the addition of oligomycin, and maximal OCR was calculated
by subtracting the nonmitochondrial OCR from the peak OCR following FCCP injection. SRC was calculated by subtracting basal OCR from max OCR. (B and E) The
ECAR over time was measured at a basal level and after the injection of oligomycin, FCCP, antimycin A, and rotenone or the injection of glucose, oligomycin, and
2-DG. Basal ECAR was determined before the addition of oligomycin, and maximal ECAR was assessed after the addition of antimycin A and rotenone. Glycolysis was
determined following the addition of glucose, and the glycolytic capacity was assessed after the addition of oligomycin. Glycolytic reserve was calculated by subtracting
basal ECAR from max ECAR or by subtracting glycolysis from glycolytic capacity. (C and F) The OCR/ECAR ratio was calculated at the basal level. (G and H)
B cells with or without Hcy treatment for 24 h were stained with 2-NBDG to assess glucose uptake via flow cytometry or imaging flow cytometry. Representative
images of bright-field and 2-NBDG (green) are shown. The data shown are representative (A–G) and cumulative (H) of at least three independent experiments [n = 5
mice in each group in (A–C)]. The data are presented as the mean 6 SEM. *p , 0.05 compared with the control. ns, not significant; Oligo, oligomycin.
glycolysis and oxidative phosphorylation are increased in Hcytreated B cells in vivo and in vitro, and that these cells show a
tendency for switching to glycolysis.
Furthermore, we incubated splenic B cells from control and
HHcy mice with LPS for an additional 24 h to test the metabolic
changes caused by stress ex vivo. HHcy-treated B cells displayed a
The Journal of Immunology
higher OCR (174.6 6 7.1 versus 139.3 6 8.3 pmol/min for basal
OCR, 304.8 6 19.5 versus 220.8 6 19.1 pmol/min for maximal
OCR, 130.2 6 16.0 versus 81.5 6 13.1 pmol/min for SRC) and
ECAR (8.7 6 0.8 versus 6.2 6 0.4 mpH/min for basal ECAR,
16.09 6 2.3 versus 11.2 6 0.8 mpH/min for max ECAR, 9.4 6
1.2 versus 5.0 6 0.5 mpH/min for glycolytic reserve) than did
control B cells (Supplemental Fig. 1A, 1B). The OCR/ECAR ratio
was not changed in HHcy-treated B cells in response to LPS
(Supplemental Fig. 1C). Flow cytometric analysis showed that
glucose uptake was increased by ∼30% in HHcy-treated B cells
(Supplemental Fig. 1D). Thus, HHcy can increase metabolic potential in B cells under both basal and stress conditions.
Distinct metabolomic profiling of Hcy-stimulated B cells
Glucose metabolism is critical for Hcy-induced B cell
activation
As revealed by the metabolic flux analysis and metabolomic
profiles, glucose metabolism is activated in Hcy-stimulated B cells
and may be critical for Hcy-induced B cell activation. To investigate the causal relationship between glucose metabolism and
Hcy-induced B cell activation, we used 2-DG to inhibit the first step
of glucose metabolism in the glycolytic pathway. As expected,
2-DG (0.5 mM) sharply suppressed Hcy-induced B cell proliferation and differentiation to plasma cells (Fig. 4A, 4B). Moreover,
2-DG (0.5–5 mM) inhibited the IgM and IgG secretion promoted
by Hcy in a dose-dependent manner (Fig. 4C, 4D). Therefore,
glucose metabolism is critical for Hcy-induced B cell proliferation
and Ab secretion.
HHcy increases PKM2 expression and enzyme activity in
B cells
The glycolytic pathway, which has many enzyme-catalyzed reactions, is the initial and major pathway in metabolizing glucose.
Pyruvate kinase is a rate-limiting glycolytic enzyme and controls
the last step of this pathway (13). Although PKM2 has been
reported to be mainly expressed in tumor cells and embryonic
cells (13, 14), recent reports showed that PKM2 is the main pyruvate kinase isoform expressed in the mouse spleen (20, 22). To
test whether B cells, one of the major cell types in the spleen,
express PKM2, we first measured the mRNA and protein levels of
the pyruvate kinase isoforms expressed in B cells. The results
showed that B cells expressed mainly PKM2 but not PKM1, as
detected by Western blotting (Fig. 5A), and by RT-PCR with
amplicons digested with restriction enzymes to distinguish Pkm1
and Pkm2 (Fig. 5B). We then sought to explore the role of PKM2
in B cells activated by HHcy. PKM2 protein expression was upregulated in B cells from HHcy mice by 50% relative to control
B cells (Fig. 5C). Moreover, the glycolytic enzyme activity of
PKM2 was also increased in HHcy-treated B cells (Fig. 5D),
suggesting a possible role of PKM2 in regulating metabolic
changes in B cells. In agreement with this, our in vitro experiments revealed that Hcy stimulation for 24 h directly upregulated
PKM2 protein expression (Fig. 5E) as well as enzyme activity
(Fig. 5F) in B cells, although gene expression of Pkm2 remained
unchanged (Fig. 5G). Therefore, PKM2 protein expression and
enzyme activity in B cells are positively regulated by HHcy both
in vivo and in vitro.
Akt-mTOR signaling pathway regulates PKM2 enzyme activity
and mediates Hcy-induced B cell activation
The Akt-mTOR signaling pathway is a critical pathway that influences cellular metabolism in immune cells through regulating
gene transcription and the posttranslational modification of glycolytic enzymes (6). Therefore, we determined whether PKM2
was modulated by the Akt-mTOR pathway in B cells upon Hcy
stimulation. Our results show that Hcy activated the Akt-mTOR
pathway by increasing the phosphorylation of Akt (Fig. 6A) and
mTOR (Fig. 6B) in B cells. We then used the mTOR inhibitor
rapamycin to explore the role of the mTOR pathway in the regulation of PKM2. Treatment with a low dose of rapamycin
(10 nM) strongly suppressed phosphorylation of mTOR and its
downstream target S6RP in Hcy-treated B cells (Fig. 6C). Although the protein expression of PKM2 was not affected
(Fig. 6D), the PKM2 enzyme activity was greatly diminished by
rapamycin in Hcy-induced B cells (Fig. 6E). PKM2, therefore, can
be one of the potential targets regulated by the mTOR pathway.
Because previous reports have established that B cell growth and
proliferation require the activation of the Akt-mTOR pathway
(12), we next determine whether the Akt-mTOR pathway is necessary for Hcy-induced B cell activation. Our results showed that
Hcy-induced B cell proliferation was partially repressed by
rapamycin (Fig. 6F). Furthermore, rapamycin also reversed Hcyinduced IgM and IgG secretion (Fig. 6G, 6H). Collectively, these
results point to a critical role for the Akt-mTOR pathway in
regulating Hcy-induced B cell activation. That PKM2 could be
regulated by the mTOR pathway indicates that PKM2 might serve
as a possible regulator in Hcy-induced B cell activation.
PKM2 mediates Hcy-induced B cell proliferation and Ab
secretion in vitro
Pyruvate kinase is a key enzyme in the glycolytic pathway that
converts phosphoenolpyruvate and ADP to pyruvate and ATP
(Fig. 7A). To verify the potential role of PKM2 in regulating Hcyinduced metabolic changes and B cell activation, the potent PKM2
inhibitor SKN was used. SKN can selectively inhibit the glycolytic enzyme activity of PKM2 without affecting other pyruvate
isoforms and may limit the subsequent lactate production and
oxidative metabolism in mitochondria (30). We first performed a
dose titration of SKN to determine the optimal dose for our experiments. Although lower doses of SKN (0.1, 0.15, 0.2 mM) only
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The extracellular flux analysis identified metabolic changes in Hcyactivated B cells. To further explore the detailed changes in
metabolic pathways and the potential underlying mechanisms
involved, metabolites were extracted from B cells stimulated with
or without Hcy for 24 h and analyzed. All samples were profiled
using LC-MS/MS. The heat map generated from hierarchical
clustering and a principal component analysis of metabolites
revealed a distinct metabolic profile in B cells upon Hcy stimulation
relative to the control group (Fig. 3A, 3B). The heat map also
showed an enrichment of metabolites in Hcy-treated B cells.
Further analysis demonstrated that Hcy-induced B cells had significantly higher levels of several glycolytic intermediates
(Fig. 3C), including GADP and 3-phosphoglycerate (3PG). The
levels of many metabolites in the TCA cycle (aconitate, isocitrate,
a-ketoglutarate, and succinyl-CoA) were enriched in B cells following Hcy stimulation (Fig. 3C), which is in line with our observations of metabolic changes in the extracellular flux analysis.
Moreover, the pentose phosphate pathway, a shunt from the glycolytic pathway, was activated, as determined by the accumulation
of xylulose 5-phosphate, sedoheptulose 7-phosphate, and NADPH
(Fig. 3C). The pentose phosphate pathway, a well-known biosynthetic pathway, is assumed to provide biosynthetic substrates to
support B cell growth and activation (12, 29). Thus, our metabolomic profiling analysis not only confirms a metabolic upregulation in both glycolytic and oxidative metabolism, but it also
suggests the activation of the biosynthesis-related pentose phosphate pathway in Hcy-treated B cells.
175
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METABOLIC REPROGRAMMING IN Hcy-INDUCED B CELL ACTIVATION
slightly impaired PKM2 activity in Hcy-treated B cells, 0.25 mM
SKN showed a significant inhibitory effect. Although a higher
dose of SKN (0.5 mM) also effectively reduced PKM2 activity, the
enzyme activity was lower than that of the control group (Fig. 7B).
To determine the contribution of Hcy-induced PKM2 enzyme
activity, a dose of 0.25 mM SKN was used in our further experiments. Although Hcy-enhanced PKM2 enzyme activity was effectively reduced, SKN did not affect PKM2 protein expression
(Fig. 7B, 7C). Next, we measured the metabolic state in B cells in
response to PKM2 inhibition. Notably, both OCR and ECAR,
which were upregulated by Hcy, were significantly reduced by
SKN (49.9% for basal OCR, 50.2% for maximal OCR, and 50.4%
for SRC, 26.9% for glycolysis, and 25.0% for glycolytic capacity;
Fig. 7D, 7E), suggesting a critical role of PKM2 in Hcy-induced
glycolytic and oxidative metabolism in B cells. Through the inhibition of metabolic changes, SKN eventually reversed Hcyinduced B cell proliferation (Fig. 7F), differentiation to plasma
cells (Fig. 7G), and Ab production (Fig. 7H, 7I). To further confirm the role of PKM2 in Hcy-induced B cell function, we
knocked down PKM2 in B cells using siRNA. Similar to SKN,
titration of PKM2 siRNA was performed and 50 nM was chosen as
the optimal dose, as this dose of PKM2 siRNA effectively re-
stricted Hcy-induced PKM2 activity without affecting the basal
PKM2 activity (Fig. 7J). PKM2 was successfully knocked down
as shown by the Western blot analysis (Fig. 7K). Similar results
were obtained, as Hcy-induced B cell differentiation and Ab secretion were reduced by PKM2 silencing (Fig. 7L–N). Taken together, these results indicate that PKM2 plays an important role in
Hcy-induced B cell activation.
In addition to Hcy stimulation, we also tested the role of PKM2
in B cells upon LPS stimulation to compare similarities and differences between Hcy and this classical stimulus of B cell activation. LPS (5 mg/ml) induced both the mRNA and protein
expression of PKM2 in B cells, whereas Hcy (100 mM) only increased PKM2 protein expression (Supplemental Fig. 2A, 2B).
B cells responded to LPS with increased PKM2 enzyme activity,
the induced level of which was relatively lower than that of the
Hcy-induced one (Supplemental Fig. 2C). SKN (0.25 mM) effectively diminished both Hcy- or LPS-induced PKM2 activity
without affecting PKM2 protein expression (Supplemental Fig.
2B, 2C). Note that the same dose of SKN reduced LPS-induced
PKM2 activity to a much lower level compared with the Hcy plus
SKN group. We then measured oxidative and glycolytic metabolism in B cells, and similar changing patterns as those in PKM2
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FIGURE 3. Distinct metabolomic profiles of Hcy-stimulated B cells. (A–C) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or
without 100 mM Hcy for 24 h. Metabolites were then extracted from three replicate B cell samples and analyzed using an LC-MS/MS system to determine
the abundance of cellular metabolites. Metabolites were analyzed using a heat map generated from hierarchical clustering (A) and a principal component
analysis (B) using MetaboAnalyst software. (C) Relative levels of metabolites in the glycolytic, pentose phosphate, and TCA cycle pathways are shown. The
data shown are representative of three independent experiments. The data are presented as the mean 6 SEM from triplicate samples. *p , 0.05 compared
with the control. DHAP, dihydroxyacetone phosphate; FBP, fructose-1,6-bisphosphate; F6P, fructose-6-phosphate; GADP, glyceraldehyde-3-phosphate;
G6P, glucose-6-phosphate; PEP, phosphoenolpyruvate; 3PG, 3-phosphoglycerate.
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177
FIGURE 4. Glucose metabolism is critical
for Hcy-induced B cell activation. (A–D)
Splenic B cells purified from C57BL/6J mice
were pretreated with or without 0.5 mM or indicated doses of 2-DG for 30 min and were then
cultured in the presence or absence of 100 mM
Hcy for the indicated times. Proliferation of
CFSE-labeled B cells (A) and CD138+ plasma
cells (B) were identified via flow cytometry at
48 h. IgM (C) and IgG (D) levels in the culture
supernatants were quantified via ELISA at 72 h.
The data shown are representative of at least
three independent experiments. The data are
presented as the mean 6 SEM. *p , 0.05
compared with the control. #p , 0.05 compared
with the Hcy group.
SKN inhibits B cell activation through regulating PKM2 and
ameliorates HHcy-accelerated atherosclerosis
We have previously reported that HHcy accelerates early atherosclerotic lesions in ApoE2/2 mice (5). Recent studies have also
reported that B cells can be tightly associated with the development of atherosclerosis (31). Given the capability of the PKM2
inhibitor SKN to prevent Hcy-induced B cell proliferation and Ab
production in vitro, we examined whether SKN could affect B cell
metabolism and function in vivo and further tested the effects of
PKM2 inhibition in HHcy-accelerated atherosclerosis in ApoE2/2
mice. ApoE2/2 mice were i.p. injected with 1.2 mg/kg SKN or
solvent control every 3 d. Three days after the first SKN injection,
ApoE2/2 mice were fed a normal mouse chow diet and given
drinking water supplemented with or without 1.8 g/l Hcy for 2 wk
(Fig. 8A). Two weeks later, a significant increase in plasma Hcy
level was detected both in HHcy mice and HHcy plus SKN mice
(Supplemental Fig. 3A), indicating that SKN treatment does
not directly affect plasma Hcy levels. Additionally, no differences were found in body weight or plasma total triglycerides
and total cholesterol levels among the four groups (Supplemental
Fig. 3B, 3C).
However, treatment with SKN greatly restored the number of
B cells in the spleen, which was increased by HHcy in ApoE2/2
mice (Fig. 8B). Notably, whereas plasma IgM levels were only
slightly reduced by SKN (Fig. 8C), SKN treatment significantly
repressed plasma IgG production in HHcy plus SKN mice relative
to production in HHcy mice (Fig. 8D). These results indicate that
SKN inhibits HHcy-induced B cell function in vivo. Additionally,
B cells from ApoE2/2 mice were collected to perform metabolicrelated experiments. Consistent with our in vitro results, PKM2
enzyme activity was enhanced in HHcy-treated B cells and was
significantly inhibited by SKN treatment (Fig. 8E). Extracellular
flux analysis revealed that the Hcy-induced elevations in both
OCR and ECAR were reversed in HHcy plus SKN mice (Fig. 8F,
8G). Furthermore, SKN treatment in vivo ameliorated HHcyaccelerated atherosclerotic lesion formation in aortic roots
(Fig. 8H). Gene expression of local inflammatory cytokines in
isolated aortas, including Icam-1, Tnf-a, Ifn-g, and Il-2, was significantly increased in HHcy ApoE2/2 mice, and this increase was
reversed by SKN treatment (Fig. 8I). Plasma inflammatory cytokines were also examined via a cytometric bead array analysis,
and no significant changes were found among the groups
(Supplemental Table I). Taken together, these results provide evidence that SKN can directly diminish HHcy-activated B cell
function in vivo via the inhibition of PKM2 enzyme activity and
metabolic changes. Moreover, SKN treatment can ameliorate
HHcy-accelerated atherosclerosis.
Discussion
Accumulating evidence demonstrates that HHcy is an independent
risk factor for cardiovascular diseases (1). As described in our
previous studies, Hcy stimulation in vitro and ex vivo can lead to
B cell proliferation and IgG secretion (3, 4). In this study, we
further verified the effects of HHcy on B cells in vivo and to our
knowledge performed the first study to explore the metabolic reprogramming and potential mechanisms involved in this
process.
The Warburg effect, or aerobic glycolysis, which is characterized
by increased lactate production rather than oxidative phosphorylation, promotes the accumulation of intermediate metabolites for
biosynthetic pathways responsible for cell growth and function
(32). Because previous reports have demonstrated that aerobic
glycolysis is critical for the proliferation and growth of tumor
cells, numerous studies have been carried out to identify whether
aerobic glycolysis is essential for innate and adaptive immunity.
Surprisingly, activated dendritic cells, M1 macrophages, and
effector T cells share some of the same properties as tumor cells,
switching their metabolic program from oxidative phosphorylation
to aerobic glycolysis upon activation, whereas resting dendritic
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activity were seen in both OCR and ECAR analysis (Supplemental
Fig. 2D, 2E). Similar results were also obtained when we examined B cell activation, as LPS induced B cell proliferation, differentiation to plasma cells, and Ab secretion, the induced levels
of which were relatively lower compared with Hcy group, whereas
most of the LPS-induced effects were also reduced by SKN, to a
greater extent than Hcy-induced effects (Supplemental Fig. 2F–I).
Collectively, these data indicate a similar role of PKM2-dependent
metabolic changes in B cells responded to LPS and Hcy. Although
the underlying mechanisms regulating PKM2 expression may
differ, these results further confirm a critical role of PKM2 in
B cell activation.
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METABOLIC REPROGRAMMING IN Hcy-INDUCED B CELL ACTIVATION
cells, M2 macrophages, and naive and memory T cells maintain
oxidative phosphorylation for energy generation (6, 8, 9). A series
of studies have revealed that B cells can be either proatherogenic
or atheroprotective, which may be dependent on the specific
context these cells reside in and on different subsets of B cells
(31). However, B cell metabolism is much less studied.
Until recently, two reports have suggested that BCR- or LPSactivated B cells and IgA+ plasma cells adopt both glycolysis
and oxidative phosphorylation to maintain cell growth and function (10, 11). Consistent with these findings, our present study
shows that Hcy stimulation causes an increase in both glycolytic
and oxidative metabolic rates in B cells. Interestingly, our in vivo
study reveals that HHcy-treated B cells, an in vivo model of
chronic Hcy stimulation, preferentially use glycolysis over oxidative phosphorylation, whereas acute (24 h) Hcy stimulation
in vitro leads to a slight decrease in the OCR/ECAR ratio without
significant changes, reflecting a more balanced use of glycolytic
and oxidative metabolism in response to an acute stimulation.
Moreover, both OCR and ECAR are more dramatically upregulated in HHcy-treated B cells after 24 h of LPS stimulation relative
to the control cells, implying a higher metabolic potential in
HHcy-treated B cells under metabolic stress.
Our metabolomic profiling analysis further confirms the increased
metabolic flux in both glycolysis and the TCA cycle. Although the
extracellular lactate production detected using the extracellular flux
analyzer was significantly increased upon Hcy stimulation, the intracellular level of lactate was not significantly altered. We speculate
that Hcy-activated B cells may swiftly export lactate produced by
glycolysis to maintain intracellular pH homeostasis. Moreover, the
pentose phosphate pathway is activated in B cells treated with Hcy, as
evidenced by the accumulation of the immediate metabolites in this
pathway. The activation of all these pathways generates substantial
energy and substrate supplies for biosynthesis to support Hcy-induced
B cell growth, proliferation, and Ab secretion.
Glucose metabolism is critical for B cell growth and activation
(12). There are many glycolytic enzymes in the glycolytic pathway; however, whether these enzymes regulate B cell function
remains largely unknown. Pyruvate kinase is a rate-limiting enzyme that converts phosphoenolpyruvate and ADP to pyruvate and
ATP. Previous studies of PKM2 mainly focused on tumor cells and
have established an important role of PKM2 in regulating tumor
cell growth through aerobic glycolysis (14). Recent reports have
indicated that PKM2 is critical for modulating macrophage activation and function (18, 19), revealing a potential role of PKM2 in
immune cells. In this study, we focused on B cells and provide
evidence that PKM2 is essential for B cell proliferation and Ab
secretion both in vivo and in vitro. B cells activated by Hcy increase their expression and enzyme activity of PKM2, as well as
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FIGURE 5. HHcy increases PKM2 expression and enzyme activity in B cells. (A) Western blot analysis with the indicated Abs was used to determine the
protein expression of pyruvate kinase isoforms in B cells. Protein from muscle was loaded as a positive control for PKM1 expression. GAPDH served as an
internal control. (B) Gene expression of total Pkm and pyruvate kinase isoforms in B cells was analyzed via RT-PCR, followed by digestion with NcoI (N), PstI
(P), or an uncut control (U). Bands are detailed as follows: total Pkm1/2, uncut (511 bp); Pkm2, PstI-cleaved fragments (364 plus 98 plus 49 bp); and Pkm1,
PstI-cleaved fragments (413 plus 98 bp) and NcoI-cleaved fragments (421 plus 84 plus 6 bp). Muscle was loaded as a positive control for Pkm1 expression. (C
and D) Splenic B cells were purified from control or HHcy C57BL/6J mice. (E–G) Splenic B cells purified from C57BL/6J mice were cultured in vitro with or
without 100 mM Hcy for 24 h in (E) and (F) or for the indicated times in (G). Cells were analyzed for PKM2 protein expression (C and E) via Western blotting
and PKM2 enzyme activity (D and F) using an LDH-coupled enzyme assay. (G) Gene expression of Pkm2 was measured via quantitative PCR in B cells at the
indicated times. The data shown are representative [(A–D); upper panel in (E)] and cumulative [lower panel in (E); (F and G)] of at least three independent
experiments [n = 3–4 mice in each group in (C and D)]. The data are presented as the mean 6 SEM. *p , 0.05 compared with the control.
The Journal of Immunology
179
their glucose uptake. Our findings that both extracellular lactate
production and oxygen consumption are markedly increased in
B cells treated with Hcy indicate that PKM2, a common upstream
glycolytic enzyme of lactate production and the TCA cycle, may
be responsible for the metabolic changes in B cells.
SKN, a potent PKM2 inhibitor, which has previously been
reported to limit glycolysis in tumor cells and protect mice from
sepsis through the inhibition of PKM2 in macrophages (18, 30),
was used in this study. Our in vitro and in vivo experiments
demonstrate that SKN inhibits the Hcy-induced upregulation of
PKM2 enzyme activity and metabolic reprogramming, leading to
the prevention of Hcy-induced B cell proliferation and Ab secretion. Although SKN shows potent inhibitory effects on PKM2,
SKN also possesses additional pharmacological effects (33). As an
additional approach, we used siRNA to knock down PKM2 and
found that Hcy-induced B cell functions are effectively inhibited,
further demonstrating a critical role of PKM2 in B cell activation.
The enhanced glycolytic function of PKM2 facilitates more glucose conversion into pyruvate and the subsequent activation of
lactate production and mitochondrial oxidative phosphorylation.
This altered metabolic phenotype allows B cells to quickly accumulate metabolites and to produce ATP simultaneously for
B cell growth and function. However, in addition to acting as a
traditional glycolytic enzyme, PKM2 also might have alternative
functions independent of enzyme activity (34). Whether PKM2
exerts nonglycolytic functions in B cells and whether these
functions are involved in B cell activation remain to be further
investigated. In addition to Hcy-activated B cells, B cells that
responded to LPS stimulation were also studied. Our data reveal a
similar role of LPS in regulating PKM2-dependent metabolic
reprogramming and B cell activation. However, differences were
seen in the underlying mechanisms regulating PKM2 expression.
LPS may increase PKM2 protein expression directly through
gene transcription as evidenced by the increased mRNA level of
Pkm2, whereas Hcy, which does not affect Pkm2 gene expression, may likely regulate this process through posttranslational
modification.
The Akt-mTOR signaling pathway is a key regulator of glucose
metabolism (35). Activation of this pathway can promote glycolysis
through altered gene transcription as well as posttranslational modifications (6). It has been reported that the increases in glucose
metabolism and cell growth in B cells activated by anti-IgM can be
effectively reversed by the inhibition of this pathway (12). In
agreement with previous findings, we demonstrate in the present
study that the Akt-mTOR pathway is critical for Hcy-induced B cell
proliferation and Ab secretion. Our findings further reveal that PKM2
could be one of the targets regulated by mTOR pathway, as PKM2
activity was limited by rapamycin treatment. However, whether
PKM2 is directly regulated by mTOR, or by the downstream targets
of the mTOR pathway, still needs further investigation.
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FIGURE 6. Akt-mTOR signaling pathway regulates PKM2 enzyme activity and mediates Hcy-induced B cell activation. (A and B) Western blot analysis and
quantification of p-AKT, AKT, p-mTOR, and mTOR protein expression in B cells treated with 100 mM Hcy for 24 h. Eukaryotic translation initiation factor 5
served as an internal control. (C–H) Splenic B cells purified from C57BL/6J mice were preincubated with 10 nM rapamycin (Rapa) for 30 min, followed by
stimulation with or without 100 mM Hcy for the indicated times. Western blot analysis of p-mTOR, mTOR, p-S6RP (C), PKM2 protein expression (D), PKM2
enzyme activity (E) at 24 h and cell proliferation (F) at 48 h were measured in B cells. IgM (G) and IgG (H) levels in culture supernatants at 72 h were measured
via ELISA. The data shown are representative [upper panels in (A–D) and (E–H)] and cumulative [lower panels in (A–D)] of at least three independent experiments. The data are presented as the mean 6 SEM. *p , 0.05 compared with the control. #p , 0.05 compared with the Hcy group. ns, not significant.
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METABOLIC REPROGRAMMING IN Hcy-INDUCED B CELL ACTIVATION
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FIGURE 7. Inhibition of PKM2 reverses Hcy-induced B cell proliferation and Ab secretion in vitro. (A) Schematic showing the rate-limiting step
regulated by PKM2 in the glycolytic pathway and the inhibition of PKM2 by SKN. (B–I) Splenic B cells purified from C57BL/6J mice were preincubated
with 0.25 mM or indicated doses of SKN for 30 min and then cultured with or without 100 mM Hcy for the indicated times. (J–N) Splenic B cells purified
from C57BL/6J mice were transfected with scramble or PKM2 siRNA. They were then treated with or without 100 mM Hcy for 48 h. Measurements of
PKM2 enzyme activity in B cells [at 24 h in (B) and at 48 h in (J)]. Cell lysates were used to measure PKM2 protein expression [at 24 h in (C) and 48 h in
(K)] via Western blot analysis. (D and E) Basal and maximal OCR, SRC, glycolysis and glycolytic capacity at 24 h were analyzed via extracellular flux
analysis. Cell proliferation (F), CD138+ plasma cells at 48 h (G and L), IgM [at 72 h in (H) and at 48 h in (M)], and IgG [at 72 h in (I) and at 48 h in (N)]
levels in culture supernatants are shown. The data shown are representative [left panels in (C) and (K); (D–I) and (L)] and cumulative [right panels in (C) and
(K); (B), (J), (M) and (N)] of at least three independent experiments. The data are presented as the mean 6 SEM. *p , 0.05 compared with the control or
scramble siRNA control. #p , 0.05 compared with the Hcy group or scramble siRNA plus Hcy group. ns, not significant.
Although our present study mainly focused on glucose
metabolism in B cells, lipid metabolism may also be involved.
Recent reports have shown that fatty acid oxidation is markedly
downregulated (10), whereas glucose-dependent de novo lipogenesis is increased in LPS-activated B cells (36). This increased
lipogenesis may support membrane expansion of the endoplasmic
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FIGURE 8. SKN inhibits B cell activation through regulating PKM2 and ameliorates HHcy-accelerated atherosclerosis. (A) Schematic flowchart of HHcy induction
and SKN treatment in ApoE2/2 mice. HHcy was induced by giving mice drinking water supplemented with 1.8 g/l Hcy. Mice were i.p. injected with 1.2 mg/kg SKN
every 3 d for the SKN treatment. Mice were divided in four groups: control (C), HHcy, SKN, and HHcy plus SKN. (B) Total numbers of splenic B cells purified from
ApoE2/2 mice were counted. (C and D) Plasma IgM and IgG levels were measured via ELISA. (E) Splenic B cells from four groups were lysed and used to assess
PKM2 enzyme activity using an LDH-coupled enzyme assay. (F and G) Time courses of OCR and ECAR in B cells are shown. Basal and maximal OCR and ECAR
were analyzed via extracellular flux analysis. (H) Oil Red O staining of aortic roots (upper panel) isolated from control and HHcy mice with or without SKN treatment.
Quantification of the mean atherosclerotic lesion area (lower panel) is shown. (I) Gene expression of Icam-1, Vcam-1, Tnf-a, Ifn-g, Mcp-1, and Il-2 in thoracic aortas
isolated from mice was measured via quantitative PCR. The data shown are representative (B–H) and cumulative (I) of at least two independent experiments (n = 3–5
mice in each group). The data are presented as the mean 6 SEM. *p , 0.05 compared with the control. #p , 0.05 compared with the HHcy group.
reticulum and Golgi network that can influence Ab synthesis and
secretion in B cells (36). These observations highlight the importance of lipid and cholesterol homeostasis in lymphocyte activation
and function. Our previous study has shown that liver X receptor
activation inhibits Hcy-induced IgG secretion in B cells (4). Liver X
receptor activation may influence many target genes involved in
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METABOLIC REPROGRAMMING IN Hcy-INDUCED B CELL ACTIVATION
defined, our present study and this most recent report consistently
highlight an important role of cellular metabolism in the regulation
of atherosclerosis and support the notion that limiting the rate of
glycolytic and oxidative metabolism by targeting PKM2 can be a
possible therapeutic approach to prevent atherosclerosis.
Collectively, our present study reveals an essential role for
PKM2-dependent metabolic reprogramming in Hcy-induced B cell
activation and function. Targeting PKM2 may provide a possible
therapeutic approach for HHcy-related atherosclerosis and B cell–
related inflammatory diseases.
Disclosures
The authors have no financial conflicts of interest.
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cholesterol and fatty acid metabolism, indicating a possible role of
lipid metabolism in Hcy-induced B cell function. These possibilities
should be thoroughly explored in future investigations.
The role of B cells and Igs in atherosclerosis remains controversial. Earlier studies have suggested an atheroprotective role for
B cells (37, 38). However, two recent studies have revealed that
the development and progression of atherosclerosis are suppressed
using a CD20-targeted B cell depletion therapy in ApoE2/2 and
low-density lipoprotein receptor–deficient mice, respectively (39,
40). These findings suggest a more complex role for B cells in
atherosclerosis, which may be dependent on the specific context
the cells reside in and on different subsets of B cells. Activated
B cells secrete Abs to maintain humoral immunity. Although IgM
is currently suggested to act as a protective role in atherosclerosis,
IgG Abs have been suggested to be either proatherogenic or
atheroprotective (31). However, there is still a lack of direct evidence for the role of Igs in atherosclerosis.
Despite these controversial findings, our results presented in this
study show that HHcy increases the number of splenic B cells,
elevates the level of plasma IgG, and increases atherosclerotic
lesion formation, which can be all restored by SKN treatment,
indicating a tight association between B cells and the development
of atherosclerosis in ApoE2/2 mice. Although blood plasma levels
of inflammatory cytokines remain unchanged, local aortic inflammatory cytokine levels are upregulated in HHcy ApoE2/2
mice and are restored by SKN treatment. Changes in local aortic
inflammatory cytokines, including Tnf-a, Il-2, and Ifn-g, secreted
by macrophages and T cells, which are in line with our previous
reports (26), suggest a role of macrophages and T cells in HHcyaccelerated atherosclerosis, and that macrophages and T cells may
also be targeted by SKN in vivo. Collectively, these results indicate that many different cell types, including B cells, T cells, and
macrophages, might together contribute to the development of
HHcy-accelerated atherosclerosis. Further investigations are
needed to confirm the exact role of B cells and Igs in this context.
Although it has been extensively established that metabolic
reprogramming is indispensable for immune cell activation and
effector function (6, 8–10), the direct relevance of cellular metabolism in relationship to atherosclerosis remains largely unknown.
Aerobic glycolysis is critical for M1 macrophage activation (9)
and has been suggested to have a proatherogenic effect by driving
proinflammatory cytokine production (41). However, the overexpression of glucose transporter 1 in macrophages does not aggravate atherosclerosis in low-density lipoprotein receptor–
deficient mice (41). This unexpected result has called into question the role of cellular metabolism in atherosclerosis. A recent
investigation focusing on hematopoietic stem and multipotential
progenitor cells has shown that glucose transporter 1 deficiency
prevents hematopoietic stem and multipotential progenitor cell
proliferation, myelopoiesis and the recruitment of Ly6Chi monocytes in atherosclerotic lesions, and eventually reduces atherosclerosis in ApoE2/2 mice (42). Actually, the most recent report
on this topic has demonstrated an overexpression of PKM2 in ex
vivo–generated macrophages collected from patients with coronary artery disease compared with expression in those from
healthy controls and has shown that PKM2 is required for M1
polarization and proinflammatory cytokine secretion from these
coronary artery disease macrophages (43). Our present study
shows that the PKM2 inhibitor SKN ameliorates HHcy-accelerated
atherosclerosis in ApoE2/2 mice. Our observation that the inhibition
of PKM2 with SKN reverses HHcy-induced local aortic proinflammatory cytokine secretion by macrophages may further support
the important role of PKM2 in macrophages and atherosclerosis.
Despite that the systemic effects of SKN in vivo have not been fully
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