Download Propionate stimulates pyruvate oxidation in the - AJP

Am J Physiol Heart Circ Physiol 307: H1134 –H1141, 2014.
First published August 22, 2014; doi:10.1152/ajpheart.00407.2014.
Innovative Methodology
Propionate stimulates pyruvate oxidation in the presence of acetate
Colin Purmal,1 Blanka Kucejova,2 A. Dean Sherry,2,3,6 Shawn C. Burgess,2,4 Craig. R. Malloy,2,3,5,7 and
Matthew E. Merritt2,6
1
School of Medicine, University of Texas Southwestern Medical Center, Dallas, Texas; 2Advanced Imaging Research Center,
University of Texas Southwestern Medical Center, Dallas, Texas; 3Department of Radiology, University of Texas
Southwestern Medical Center, Dallas, Texas; 4Department of Pharmacology, University of Texas Southwestern Medical
Center, Dallas, Texas; 5Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas;
6
Department of Chemistry, University of Texas at Dallas, Richardson, Texas; and 7Veterans Affairs North Texas Healthcare
System, Dallas, Texas
Submitted 12 June 2014; accepted in final form 21 August 2014
substrate selection; pyruvate; glucose; propionate; hyperpolarization;
isotopomer analysis
myocardial ischemia inhibits
production of hyperpolarized (HP) [13C]bicarbonate derived
from HP [1-13C]pyruvate (8, 9, 36). Because the appearance of
HP [13C]bicarbonate is due to metabolism in functioning mitochondria, the appearance of HP [1-13C]bicarbonate may
provide evidence of viable myocardium (3, 18, 25, 26, 35, 36).
Consequently, it is important to understand the factors controlling appearance of HP [1-13C]bicarbonate from HP [1-13C]pyruvate. Under simple conditions in isolated hearts, HP [113
C]bicarbonate is generated from HP [1-13C]pyruvate only
by flux through pyruvate dehydrogenase (PDH) (25). Flux
through this pathway is inhibited by acetyl-CoA and other end
END STAGE CARDIOMYOPATHY OR
Address for reprint requests and other correspondence: M. E. Merritt,
Advanced Imaging Research Center, Univ. of Texas Southwestern Medical
Center, Dallas, Texas 75390 (e-mail: [email protected]).
H1134
products, and further regulated by phosphorylation-dephosphorylation of the PDH complex. Because metabolism of fatty
acids generates high concentrations of acetyl-CoA, the appearance of HP 13CO2 and HP [13C]bicarbonate is markedly inhibited by fats even in normal hearts (25, 27). Consequently, in
addition to disease processes, physiological fluctuations in the
concentrations of fatty acids also influence the appearance of
HP [13C]bicarbonate. An intervention that stimulates flux
through PDH in the presence of fatty acids could remove the
effects of substrate competition in detecting oxidation of HP
[1-13C]pyruvate. One such approach is co-administration of
glucose, insulin, and potassium (16). This mixture has multiple
effects on plasma glucose, plasma free fatty acids, and myocardial membrane potential, so any effects on bicarbonate
appearance probably reflect a complex interaction between
substrate concentrations and direct effects on PDH (4).
Propionate, a physiological short chain fatty acid, also influences PDH flux. The interaction of propionate with pyruvate
metabolism in the heart has been extensively studied using 14C
tracers. Fatty acids of even carbon chain length markedly
inhibit appearance of 14CO2 from [1-14C]pyruvate (14, 32).
When compared with acetate, propionate increased the rate of
14
CO2 production from [1-14C]pyruvate under steady state
conditions (14), suggesting that propionate may be an alternative to glucose, insulin, and potassium for stimulating PDH
flux. However, the relevance of these studies for HP 13C NMR
observations is unclear, for two reasons. First, the radiotracer
studies examined the influence of individual fatty acids such as
acetate or propionate on pyruvate metabolism. The effect of
propionate on pyruvate metabolism in the presence of even
chain fatty acids has not been studied extensively, and it is not
known whether the stimulatory effects of propionate on PDH
flux overcome the inhibitory effects of fatty acids. Second, the
magnitude of the increase in 14CO2 appearance from [114
C]pyruvate induced by propionate was only about threefold
compared with acetate (14). Because these data were acquired
under steady state conditions, it is not clear if HP pyruvate
would detect similar effects, since the window for observing
the HP species is ⬃2 min.
The purpose of this study was to determine whether the
reduced appearance of HP [13C]bicarbonate from HP [113
C]pyruvate caused by acetate can be overcome by propionate-induced stimulation of PDH flux. Hearts from C57/bl6
mice were perfused to steady state in the presence of glucose
and saturating concentrations of acetate. Acetate is the most
avidly metabolized fatty acid, since it freely diffuses into the
mitochondria, bypassing carnitine palmitoyl transferase-I. In
0363-6135/14 Copyright © 2014 the American Physiological Society
http://www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
Purmal C, Kucejova B, Sherry AD, Burgess SC, Malloy CR,
Merritt ME. Propionate stimulates pyruvate oxidation in the presence of
acetate. Am J Physiol Heart Circ Physiol 307: H1134 –H1141, 2014. First
published August 22, 2014; doi:10.1152/ajpheart.00407.2014.—Flux
through pyruvate dehydrogenase (PDH) in the heart may be reduced by
various forms of injury to the myocardium, or by oxidation of
alternative substrates in normal heart tissue. It is important to distinguish these two mechanisms because imaging of flux through PDH
based on the appearance of hyperpolarized (HP) [13C]bicarbonate
derived from HP [1-13C]pyruvate has been proposed as a method for
identifying viable myocardium. The efficacy of propionate for increasing PDH flux in the setting of PDH inhibition by an alternative
substrate was studied using isotopomer analysis paired with exams
using HP [1-13C]pyruvate. Hearts from C57/bl6 mice were supplied
with acetate (2 mM) and glucose (8.25 mM). 13C NMR spectra were
acquired in a cryogenically cooled probe at 14.1 Tesla. After addition
of hyperpolarized [1-13C]pyruvate, 13C NMR signals from lactate,
alanine, malate, and aspartate were easily detected, in addition to
small signals from bicarbonate and CO2. The addition of propionate (2
mM) increased appearance of HP [13C]bicarbonate ⬎30-fold without
change in O2 consumption. Isotopomer analysis of extracts from the
freeze-clamped hearts indicated that acetate was the preferred substrate for energy production, glucose contribution to energy production was minimal, and anaplerosis was stimulated in the presence of
propionate. Under conditions where production of acetyl-CoA is
dominated by the availability of an alternative substrate, acetate,
propionate markedly stimulated PDH flux as detected by the appearance of hyperpolarized [13C]bicarbonate from metabolism of hyperpolarized [1-13C]pyruvate.
Innovative Methodology
H1135
PROPIONATE STIMULATES PYRUVATE OXIDATION
addition, it is not ␤-oxidized, but can immediately be used to
synthesize acetyl-CoA. As such, the acetate perfused heart
represents a model where flux through PDH is expected to be
maximally inhibited. In addition, to our knowledge, this is the
first application of a combination of hyperpolarization and
cryoprobe technology to study the perfused mouse heart.
MATERIALS AND METHODS
RESULTS
Effects of propionate on oxidation of acetate and glucose.
The presence of propionate in the perfusion medium did not
alter O2 consumption (44 ⫾ 21 ␮mol·gdw⫺1·min⫺1 in group 1
vs. 48 ⫾ 15 ␮mol·gdw⫺1·min⫺1 in group 2). Flux through
citrate synthase was calculated from O2 consumption (17).
Table 1 summarizes these values for the two different conditions. Table 1 also reports that anaplerotic flux (relative to TCA
cycle flux) was significantly elevated in the presence of propionate as expected. There was no significant change in phosphorylation at serine 293 of PDH as measured in Western blots
in hearts supplied with propionate (data not shown).
The effects of unlabeled propionate on conventional 13C
NMR spectra from extracts of hearts supplied with [1,213
C2]acetate plus unlabeled glucose are illustrated in Fig. 1. In
addition to the dominant glutamate resonances, the spectra also
show resonances of malate, citrate, and aspartate, with malate
and citrate being higher when propionate is supplied (Fig. 1B).
The insets in Fig. 1 show the glutamate C2, C3, and C4
resonances. Isotopomers of glutamate that have three or more
carbons labeled consecutively produce doublets of doublets,
marked Q for quartet when the J-couplings are resolved, or T
for triplet when they are degenerate as occurs in carbon 3 of
glutamate. Glutamate labeled in positions 3, 4, and 5 is derived
from the condensation of [U-13C]acetyl-CoA with [2-13C]oxaloacetate and its subsequent forward flux to ␣-ketoglutarate
and exchange into the glutamate pool.
The relative areas of the multiplets in each glutamate resonance provide a direct read-out of substrate preferences and
anaplerosis under steady-state isotopic conditions (17, 19 –21).
Table 1. Metabolic fluxes calculated from a combination of
13
C-isotopomer analysis of 13C NMR spectra of perfused
mouse heart extracts plus O2 consumption data
Without propionate
With propionate
O2 Consumption,
␮mol·gdw⫺1·min⫺1
Citrate Synthase Flux,
␮mol·gdw⫺1·min⫺1
Y
44 ⫾ 22
48 ⫾ 15
21 ⫾ 9
22 ⫾ 7
0.12 ⫾ 0.01
0.25 ⫾ 0.02
Anaplerosis (Ys) is expressed as a fraction of citrate synthase flux (TCA
cycle turnover). Anaplerosis, y, is significantly different (P ⬍ 0.001).
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00407.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
Materials. [1-13C]pyruvic acid and [2-13C]pyruvic acid were purchased from Sigma-Aldrich Isotec (Miamisburg, OH) or Cambridge
Isotope Laboratories (Tewksbury, MA) and used without further
purification. The various 13C isotopomers of sodium acetate, glucose,
and propionate were purchased and used as supplied from Cambridge
Isotopes (Andover, MA). Trityl-OXO63 free radical was purchased
from Oxford Instruments Molecular Biotools (Oxford, UK).
Protocol. The protocol was approved by the Institutional Animal
Care and Use Committee of the University of Texas Southwestern
Medical Center. Female mice, 14 –16 wk in age, were purchased from
Charles River and housed four to a cage. All animals were fed ad
libitum. A single ketamine (43 mg/kg) and xylazine (8.7 mg/kg)
intraperitoneal injection was used for general anesthesia. Depth of
anesthesia was assessed by observation of the respiratory rate, paw
pinch reflex, and palpebral reflex of the animal. Under general
anesthesia, the hearts were rapidly excised and arrested in ice-cold
perfusion medium. Langendorff perfusions were performed according
to Stowe et al. (39) including measurements of developed pressure,
coronary flow, heart rate, and O2 consumption. Briefly, the aorta of
the excised heart was cannulated and perfused in nonrecirculating
mode at a constant perfusion pressure of 80 cm H2O. A KrebsHenseleit buffered medium containing 25 mM NaHCO3, 118 mM
NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 0.5 mM
ethylene diamine tetraacetic acid was bubbled continuously with a
95/5 mixture of O2/CO2 gas to maintain a pH of 7.4. The perfusing
medium was maintained at 37°C using a water bath.
Two sets of perfusion conditions were used in the studies of HP
[1-13C]pyruvate. Hearts in group 1 were supplied with 8.25 mM
glucose and 2 mM [U-13C]acetate with subsequent addition of HP
pyruvate (n ⫽ 5). Hearts in group 2 were perfused with identical
concentrations of glucose and acetate plus 2 mM propionate (n ⫽ 5).
Additional experiments were performed with unlabeled acetate, [213
C]pyruvate, [U-13C]glucose, or [U-13C]propionate, depending on
the experiment. Hearts were freeze-clamped and extracted for 1H and
13
C NMR spectroscopy.
Dynamic nuclear polarization. All NMR experiments were carried
out at 14.1 T using a Bruker Avance 3 HD console (Bruker BioSpin,
Billerica, MA) equipped with a 10 mm 13C cryoprobe operating at a
temperature of 15° K. The temperature of the sample was held at 37°C
throughout the experiment. Samples of pyruvic acid (5 ␮l) were
polarized essentially as described previously (1). The samples were
dissolved and injected by catheter immediately above the heart (25).
13
C NMR spectra were acquired using 15° excitation pulses with a 2-s
repetition time. Acquisition was terminated upon completion of 96
single-pulse acquisitions, the perfusion rig was removed from the
magnet, and the heart was immediately freeze-clamped for tissue
extraction. The time delay between initial injection of pyruvate to
freeze-clamping, including all NMR data acquisition from the perfused heart, was ⬃200 s. Peak areas from the DNP experiments were
measured by integration.
NMR and isotopomer analysis of tissue extracts. Freeze-clamped
hearts were extracted using perchloric acid (27). 13C NMR of the
extracts was acquired as previously (24). The resulting multiplets
derived from the utilization of the isotopically labeled substrates were
fit using a mixed Gaussian/Lorentzian lineshape in the ACD NMR
Processor Software (Advanced Chemistry Development, Toronto,
Canada). Proton spectra were fitted with the Chenomx software
package to obtain lactate and alanine concentrations (Chenomx, Al-
berta, Canada). Relative peak areas of the resonances adjacent to 12C
versus the 13C satellites were measured in the 1H NMR spectrum to
obtain a final concentration of the metabolites as well as the 13C
enrichments. The 13C NMR spectra of glutamate were analyzed
according to Sherry et al. (37). Fractions of acetyl-CoA derived from
[U-13C]acetate were also analyzed using the C5D/C5S ratio.
Western blot analysis. Tissue lysates were prepared in RIPA buffer
(Cell Signaling, Danvers, MA) containing CompleteTM protease inhibitors (Roche, Indianapolis, IN). Anti-PDH ␣1 subunit antibodies
were obtained from Cell Signaling, and anti-phospho-PDH ␣1 subunit
(S293) were obtained from Millipore (Billerica, MA).
Statistical analysis. Significant differences were evaluated using
GraphPad Prism (La Jolla, CA) using multiple t-tests with a significance level of P ⬍ 0.05. For the HP data, each resonance was
analyzed individually without the assumption of a consistent standard
deviation. The False Discovery Rate approach with a rate of 5% was
used to suppress random correlations between variables. For comparisons of concentrations and fractional enrichments, simple t-tests were
used without further correction. For all graphs, error bars reflect the
standard deviation of the data.
Innovative Methodology
H1136
PROPIONATE STIMULATES PYRUVATE OXIDATION
The dominant signal from [3,4,5-13C3]glutamate C4 (the doublet of doublet, or quartet) indicates qualitatively that a high
fraction of acetyl-CoA was derived from exogenous [1,213
C2]acetate (Fig. 1A). The [3,4,5-13C]glutamate isotopomer is
decreased in the presence of propionate as evidenced by the
increase in the relative fraction of the D45 doublet versus
the C4Q fraction (Fig. 1B). This isotopomer is produced by
the condensation of [U-13C]acetyl-CoA with oxaloacetate
that is not labeled in the C2 position. Under these conditions, unenriched propionate increases the fraction of OAA
that is not labeled. The relevant pathways are summarized in
Fig. 2. The isotopomers of ␣-ketoglutarate derived from
[1,2-13C2]oxaloacetate or [3,4-13C2]oxaloacetate are shown
in the dotted box. With the addition of unlabeled propionate
and consequently an increase in the fraction of unlabeled
oxaloacetate, the fraction of ␣-ketoglutarate that is [3,4,5C]␣-ketoglutarate will decrease (Fig. 1B).
Because the heart was clamped less than 3.5 min after
addition of [1-13C]pyruvate, the 13C NMR spectrum is dominated by isotopomers generated before addition of pyruvate.
Conventional isotopomer analysis was used to measure anaplerosis and the fraction of acetyl-CoA derived from [1,213
C2]acetate (20). After the introduction of [1-13C]pyruvate,
metabolism through PDH could briefly provide a new source
of unenriched acetyl-CoA. In hearts supplied with acetate
and glucose, 98% of acetyl-CoA was derived from [1,213
C2]acetate. Among the hearts supplied with acetate, glucose,
and propionate, the same amount of acetyl-CoA was derived
from [1,2-13C2]acetate, as measured by the fraction of C5D/
C5S. The singlet was assumed to be derived from natural
13
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00407.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
Fig. 1. 1H-Decoupled 13C NMR spectra of tissue extracts. Thermally polarized 13C NMR spectra of tissue extracts of hearts perfused to steady state with unlabeled
glucose and [U-13C]acetate and exposed briefly to hyperpolarized (HP)-[1-13C]pyruvate are illustrated over the chemical shift range of ⬃25 to 57 ppm. In the
absence of propionate (A), both acetyl-CoA and oxaloacetate must be highly enriched as measured by the high fractional contribution of the quartets (in C2 and
C4) or triplets (in C3) of glutamate. Hearts supplied with propionate (B) showed an increase in the doublet, D45, relative to the quartet, indicating lower 13C
enrichment in oxaloacetate. Propionate also induced an increase in citrate and malate abundance. C: concentrations (conc.) of lactate and alanine as measured
by quantitative 1H NMR of the heart extracts. Addition of propionate to the perfusion medium resulted in a decrease in the alanine concentration in the heart.
D: fractional enrichments of the C1 position of lactate and alanine as measured by proton NMR of the methyl protons and 3 bond j-couplings to the C1 position.
Alanine is more enriched than lactate in both perfusions conditions, although the difference is accentuated in the presence of propionate.
Innovative Methodology
PROPIONATE STIMULATES PYRUVATE OXIDATION
H1137
Fig. 2. Metabolic model for hearts supplied with [1,2-13C2]acetate. Ball-andstick figures of acetate, acetyl-CoA, pyruvate, oxaloacetate (OAA), ␣-ketoglutarate, and glucose (2, 2, 3, 4, 5, or 6 carbons, respectively) are shown. 13C is
indicated by filled circles, and each carbon position is numbered from left to
right. The isotopomers presented assume 1 turn of the TCA cycle has been
completed with labeled acetate present, hence the symmetric enrichment of the
OAA pool. Under these conditions, neglecting natural abundance 13C, the
acetyl-CoA pool is either [1,2-13C2]acetyl-CoA or unlabeled. Consequently,
the C4 signal of glutamate is either a doublet due to labeling in carbons 4 and
5, or a doublet of doublets due to labeling in positions 3 and 4 and 5. In the
presence of propionate, the fraction of unenriched OAA is increased and,
consequently, the fraction of ␣-ketoglutarate labeled simultaneously in carbons
3 and 4 and 5 decreases. The dashed rectangle indicates ␣-ketoglutarate
derived from 13C-labeled OAA. PDH, pyruvate dehydrogenase.
abundance 13C. Separate bench experiments using [U-13C]glucose and [2-13C]acetate confirmed that acetate was the overwhelming source of acetyl-CoA; less than 3% of acetyl-CoA
was derived from glucose either in the presence or absence of
propionate. These experiments confirmed that exogenous glucose was not metabolized in the TCA cycle via either PDH or
pyruvate carboxylase, consistent with earlier studies of isolated
rat hearts (20).
1
H NMR spectra of these same samples indicated that
propionate resulted in a significant decrease in total tissue
alanine and a trend toward lower lactate (Fig. 1, C and D). The
lactate-to-alanine ratio was not significantly different. Similarly, the fractional 13C enrichment in the lactate was not
significantly different in the presence of propionate, but the
enrichment in [1-13C]alanine was significantly higher in propionate-perfused hearts. Labeling in the C1 position can only
result from the exogenous, hyperpolarized [1-13C]pyruvate.
Fig. 3. Metabolism of [1-13C]pyruvate. Flux through PDH leads to production
of 13CO2, but there are 3 other possible sources in a single turn of the TCA
cycle. After carboxylation by PC, the 13C label can become symmetrized by
exchange between malate and fumarate (faded circle). Subsequent flux through
the malic enzyme produces 13CO2 but the fractional enrichment has a maximal
value of 50%. Alternatively, after carboxylation, a forward turn of the TCA
cycle could eliminate a 13CO2 at the step of isocitrate dehydrogenase (shaded
circle). This pathway could have a maximal enrichment equal to the exogenous
pyruvate. Finally, after symmetrization at fumarate, a forward turn of the TCA
cycle could produce a 13CO2 at ␣-ketoglutarate dehydrogenase.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00407.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
Both [1-13C] and [2-13C]lactate and alanine have similar 2JCH
and 3JCH values to the methyl protons, but inspection of the 13C
spectra allow the couplings to be assigned exclusively to
labeling at the C1 position. This provides direct evidence
showing that exogenous [1-13C]pyruvate exchanges more fully
with tissue pools of alanine than it does with tissue lactate, as
reported previously (31).
Effect of propionate on metabolism of hyperpolarized
[1-13C]pyruvate. Mouse hearts were studied in the 10 mm 13C
optimized cryoprobe. All four possible biochemical fates of
[1-13C]pyruvate, illustrated in Fig. 3, could be directly detected
in the functioning mouse heart. Metabolism of pyruvate to
lactate, alanine, or bicarbonate could be monitored in real
time, and the fourth possibility - carboxylation via pyruvate
carboxylase - could be detected in summed spectra. Spectra
obtained by summing the 25 individual spectra with the
highest signal following injection with hyperpolarized [113
C]pyruvate are shown in Fig. 4. Downstream products
resulting from carboxylation of pyruvate (malate and aspartate) as well as the expected signals of [1-13C]lactate, [1-
Innovative Methodology
H1138
PROPIONATE STIMULATES PYRUVATE OXIDATION
DISCUSSION
C]alanine, and H13CO3⫺ were observed. In hearts supplied
with propionate in the perfusion media, there was a marked
increase in the signal intensity of HP [13C]bicarbonate and a
small decrease in signal intensities of aspartate and malate. The
increase in the malate signal in conventional 13C NMR spectra
shows that the pool size of malate is increased by propionate
(Fig. 1), a well-known effect (40), yet the HP malate signal was
reduced.
Representative time-intensity curves for [1-13C]lactate, [113
C]alanine, H13CO3⫺, and 13CO2 are shown in Fig. 5. The
most striking differences were in the magnitude of the [13C]bicarbonate and 13CO2 signals in the presence of propionate. An
alternative approach to display data such as this is to normalize
the total signal intensity derived from pyruvate and its metabolites and graph the hyperpolarized “activities” of each, i.e.,
taking the area under the curve for the time evolution of each
metabolite (Fig. 6) (11). The integrated [13C]bicarbonate intensity was ⬃37 times higher in the presence of propionate
(P ⬍ 0.01). Other significant differences were found for
alanine (P ⬍ 0.01), aspartate-C1 (P ⬍ 0.01), aspartate-C4 (P ⬍
0.01), and for 13CO2 (P ⬍ 0.01), which is in fast equilibrium
with bicarbonate. No significant differences were observed for
signals associated with lactate or either C1- or C4-labeled
malate.
13
Fig. 5. Effect of propionate on dynamic appearance of lactate, alanine,
bicarbonate, and CO2. Representative kinetic curves for appearance of [113
C]lactate, [1-13C]alanine, 13CO2, and [13C]bicarbonate in the absence (B) and
presence (A) of propionate after addition of HP [1-13C]pyruvate are shown.
Propionate perfusion causes not only an increase in [1-13C]pyruvate decarboxylation but also a decrease and a change in the time to maximum signal for the
[1-13C]alanine.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00407.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
Fig. 4. 13C spectra of the functioning mouse heart. Spectra were obtained
following the addition of HP [1-13C]pyruvate to perfused hearts without
propionate and with propionate. The [13C]bicarbonate ([13C]bicarb) signal was
increased in the presence of propionate, but there was little effect on the
intensity of the malate-C4 resonances. If the increase in [13C]bicarbonate
signal is derived from malic enzyme or ␣-ketoglutarate dehydrogenase flux, an
increase in the malate resonance intensity would be expected. Pyr-hyd, pyruvate hydrate.
This experiment was designed to assess the effects of propionate on pyruvate metabolism in the presence of a high
concentration of a fatty acid. Acetate was chosen because there
is no question that it is the preferred substrate for oxidation by
heart tissue when present in millimolar concentrations, and it
therefore serves as a model for maximal substrate-induced
suppression of PDH flux. These studies confirm that in the
presence of high concentrations of acetate and glucose, the
overwhelming majority of acetyl-CoA production in hearts was
derived from acetate (20). The current results further demonstrate that oxidation of HP [1-13C]pyruvate, even in a high
concentration administered as a bolus, is nearly eliminated in
the presence of acetate (27). Oxygen consumption was not
changed by propionate (Table 1), yet HP [13C]bicarbonate
increased ⬎30-fold (Fig. 5). A much smaller increase in HP
[13C]bicarbonate was noted under conditions where malate was
co-administered in vivo (34). Administration of dichloroacetate also activates PDH flux, but side effects of the drug
prevent its long-term usage medically (2, 12, 23). This model
is another condition where bicarbonate production from HP
[1-13C]pyruvate is completely dissociated from myocardial
oxygen consumption (25). Earlier studies examined the effects
of acetate or propionate, studied independently, on [1-14C]pyruvate metabolism and the rate of appearance of 14CO2 compared with the absence of either fatty acid. These studies
suggest approximately threefold increase in 13CO2 could be
expected in the presence of propionate. The current observation of ⬎30-fold increase likely occurred for two reasons. First,
more physiological concentration of pyruvate, ⬃0.1 mM, was
used previously (14). The current design delivered a much
higher concentration of pyruvate, 2 mM. Although this is
Innovative Methodology
PROPIONATE STIMULATES PYRUVATE OXIDATION
higher than the physiological concentration of pyruvate in
normal plasma, millimolar concentrations of pyruvate are common and probably necessary for in vivo HP experiments (33).
A second factor in the large effect of propionate on [13C]bicarbonate production is the control condition. In the study
design, advantage was taken of the known effects of acetate on
pyruvate oxidation and the anticipated marked suppression on
the appearance of HP [13C]bicarbonate and 13CO2 in control
hearts. The present results demonstrate that the stimulatory
effects of propionate on PDH flux override the inhibitory
effects of acetate on appearance of HP [13C]bicarbonate from
HP [1-13C]pyruvate. Acetate is not subject to transport by
carnitine palmitoyl transferase-I, nor is it ␤-oxidized. As such,
it is the most avidly metabolized substrate available to the
heart. The overwhelming preference of the heart for acetate
underscores just how powerful the effects of propionate are on
substrate selection. The activity of the PDH is regulated by
reversible phosphorylations of multiple subunits and by inhibition by its end products, acetyl CoA and NADH. The
phosphorylation state of PDH, in turn, is controlled by competing activity of pyruvate dehydrogenase kinase and pyruvate
dehydrogenase phosphatase. In the absence of acetate, propionate caused almost complete dephosphorylation of PDH in rat
hearts (14). Under the current experimental conditions, with a
high concentration of both acetate and propionate in group 2,
the Western blot analysis did not demonstrate a measurable
change in the phosphorylation state at serine 293 (data not
shown). The effect of propionate on PDH flux could be due to
trapping of CoA as propionyl-CoA rather than acetyl-CoA,
with a consequent loss of end-product inhibition at PDH (41).
Alternatively, propionate could exert control through secondary phosphorylation sites on PDH (10). Exact determination of
the mechanism of PDH activation in this condition is the
subject of ongoing research.
Pyruvate may also undergo carboxylation in the heart (20) as
illustrated in Fig. 3, which allows additional decarboxylation
reactions that could serve as sources of HP [13C]bicarbonate
(30, 38). As illustrated in Fig. 4, HP [1-13C]malate, HP [113
C]aspartate, and HP [4-13C]aspartate were all detected. Because the T1 of 13C limits the duration of these studies,
conversion of [1-13C]pyruvate ¡ [1-13C]oxaloacetate ¡ [113
C]malate ¡ [1-13C]fumarate ⫹ [4-13C]fumarate ¡ [413
C]malate ¡ [4-13C]aspartate must have occurred within 10 s
of addition of pyruvate. The presence of HP [4-13C]aspartate
demonstrates that [4-13C]oxaloacetate must also be present.
Phosphoenolpyruvate carboxykinase does not exist in the
heart, but the malic enzyme can serve to cycle malate back to
pyruvate with release of CO2 (40). Introduction of propionate
produced highly significant increases in both HP 13CO2 and
[13C]bicarbonate. The HP signals of aspartate-C1 and -C4 were
both lower in the presence of propionate, but the HP signals of
malate-C1 and -C4 only trended lower without reaching statistical significance. Given the stable or declining pool sizes of
malate and aspartate (Fig. 1), this result indicates that the
amount of [1-13C]pyruvate entering the citric acid cycle via
pyruvate carboxylation is reduced when propionate is present. By extension, the increase in anaplerosis shown by 13C
isotopomer analysis can be largely ascribed to entry of propionate into the TCA cycle via propionyl-CoA carboxylase.
Consequently, we conclude that production of 13CO2 via carboxylation of HP [1-13C]pyruvate followed re-arrangement
and eventual decarboxylation contributes little, probably insignificant amounts, to overall appearance of HP[13C]bicarbonate
under these conditions. Furthermore, if the malic enzyme was
highly active, pyruvate formed by decarboxylation of malate
would be labeled in multiple positions concurrently due to the
high fractional enrichment of the oxaloacetate pool achieved
by perfusion with [1,2-13C2]acetate. Although pyruvate is not
detectable in the extracts, the lactate and alanine pools did not
demonstrate any labeling beyond that caused by the injection
of HP [1-13C]pyruvate. Finally, in the presence of HP [213
C]pyruvate, 13C labeling observed in citrate and glutamate
could only arise from decarboxylation of HP [2-13C]pyruvate by the PDH (data not shown). Together, these spectra
indicate that the only significant source of [13C]bicarbonate
is the product of pyruvate decarboxylation in PDH. These
observations are consistent with previous findings in the rat
heart (14, 20, 38) and further demonstrate the stimulatory
effects of propionate on flux of exogenous pyruvate through
PDH even in the presence of inhibition by short chain fatty
acid metabolism.
In addition to its effects on pyruvate oxidation, exposure to
propionate also stimulates anaplerosis in the heart (5, 14). In
this case, propionate itself is the anaplerotic substrate. After
activation to propionyl-CoA and carboxylation to form methylmalonyl-CoA, it undergoes rearrangement to an intermediate
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00407.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
Fig. 6. Effect of propionate on hyperpolarized signal of metabolic products.
The integrated areas under the curve expressed as the fraction of total intensity
of pyruvate and its metabolites are shown for 8 signals. The 4-carbon
intermediates are ⬃4 – 8 times less intense than the alanine and lactate signals.
Lack of measurable differences in intensity of the malate (mal)-C4 resonance
strongly suggests the [13C]bicarbonate (bicarb) production is not related to
increased malic enzyme flux. *P ⬍ 0.05. Asp, aspartate.
H1139
Innovative Methodology
H1140
PROPIONATE STIMULATES PYRUVATE OXIDATION
GRANTS
This research was funded through National Heart, Lung, and Blood Institute
Grants R21 EB016197, P41 EB015908, R37 HL34557, and R01 DK058398.
Dr. Merritt acknowledges salary support from CPRIT RP-101243. S. C.
Burgess acknowledges support from the Robert A. Welch foundation.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: C.P., B.K., and M.E.M. performed experiments; C.P.,
A.D.S., S.C.B., C.R.M., and M.E.M. edited and revised manuscript; A.D.S.,
S.C.B., C.R.M., and M.E.M. interpreted results of experiments; C.R.M. and
M.E.M. analyzed data; M.E.M. conception and design of research; M.E.M.
prepared figures; M.E.M. drafted manuscript; M.E.M. approved final version
of manuscript.
REFERENCES
1. Ardenkjaer-Larsen JH, Fridlund B, Gram A, Hansson G, Hansson L,
Lerche MH, Servin R, Thaning M, Golman K. Increase in signal-tonoise ratio of ⬎ 10,000 times in liquid-state NMR. Proc Natl Acad Sci
USA 100: 10158 –10163, 2003.
2. Atherton HJ, Dodd MS, Heather LC, Schroeder MA, Griffin JL,
Radda GK, Clarke K, Tyler DJ. Role of pyruvate dehydrogenase
inhibition in the development of hypertrophy in the hyperthyroid rat heart:
a combined magnetic resonance imaging and hyperpolarized magnetic
resonance spectroscopy study. Circulation 123: 2552–2561, 2011.
3. Atherton HJ, Schroeder MA, Dodd MS, Heather LC, Carter EE,
Cochlin LE, Nagel S, Sibson NR, Radda GK, Clarke K, Tyler DJ.
Validation of the in vivo assessment of pyruvate dehydrogenase activity
using hyperpolarised 13C MRS. NMR Biomed 24: 201–208, 2011.
4. Bothe W, Olschewski M, Beyersdorf F, Doenst T. Glucose-insulinpotassium in cardiac surgery: a meta-analysis. Ann Thorac Surg 78:
1650 –1657, 2004.
5. Brunengraber H, Roe CR. Anaplerotic molecules: current and future. J
Inherit Metab Dis 29: 327–331, 2006.
6. Ferrari R, Merli E, Cicchitelli G, Mele D, Fucili A, Ceconi C.
Therapeutic effects of l-carnitine and propionyl-l-carnitine on cardiovascular diseases: a review. Ann N Y Acad Sci 1033: 79 –91, 2004.
7. Garland PB, Newsholme EA, Randle PJ. Regulation of glucose uptake
by muscle. Effects of fatty acids and ketone bodies, and of alloxandiabetes and starvation, on pyruvate metabolism and on lactate-pyruvate
and L-glycerol 3-phosphate-dihydroxyacetone phosphate concentration
ratios in rat heart and rat diaphragm muscles. Biochem J 93: 665–678,
1964.
8. Golman K, Petersson JS. Metabolic imaging and other applications of
hyperpolarized 13C. Academic Radiology 13: 932–942, 2006.
9. Golman K, Petersson JS, Magnusson P, Johansson E, Åkeson P, Chai
C, Hansson G, Månsson S. Cardiac metabolism measured noninvasively
by hyperpolarized 13C MRI. Magn Reson Med 59: 1005–1013, 2008.
10. Holness MJ, Sugden MC. Regulation of pyruvate dehydrogenase complex activity by reversible phosphorylation. Biochem Soc Trans 31:
1143–1151, 2003.
11. Hu S, Chen AP, Zierhut ML, Bok R, Yen YF, Schroeder MA, Hurd
RE, Nelson SJ, Kurhanewicz J, Vigneron DB. In vivo carbon-13
dynamic MRS and MRSI of normal and fasted rat liver with hyperpolarized 13C-pyruvate. Mol Imaging Biol 11: 399 –407, 2009.
12. Hu S, Yoshihara HAI, Bok R, Zhou J, Zhu M, Kurhanewicz J,
Vigneron DB. Use of hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate to probe the effects of the anticancer agent dichloroacetate on
mitochondrial metabolism in vivo in the normal rat. Magn Reson Imaging
30: 1367–1372, 2012.
13. Lango R, Smolenski RT, Rogowski J, Siebert J, Wujtewicz M, Slominska EM, Lysiak-Szydlowska W, Yacoub MH. Propionyl-L-carnitine
improves hemodynamics and metabolic markers of cardiac perfusion
during coronary surgery in diabetic patients. Cardiovasc Drugs Ther 19:
267–275, 2005.
14. Latipaa PM, Peuhkurinen KJ, Hiltunen JK, Hassinen IE. Regulation
of pyruvate dehydrogenase during infusion of fatty acids of varying chain
lengths in the perfused rat heart. J Mol Cell Cardiol 17: 1161–1171, 1985.
15. Lau AZ, Chen AP, Ghugre NR, Ramanan V, Lam WW, Connelly KA,
Wright GA, Cunningham CH. Rapid multislice imaging of hyperpolarized 13C pyruvate and bicarbonate in the heart. Magn Reson Med 64:
1323–1331, 2010.
16. Lauritzen MH, Laustsen C, Butt SA, Magnusson P, Søgaard LV,
Ardenkjær-Larsen JH, Åkeson P. Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1-13C]pyruvate MRS studies by infusion of
glucose, insulin and potassium. NMR Biomed 26: 1496 –1500, 2013.
17. Malloy CR, Jones JG, Jeffrey FM, Jessen ME, Sherry AD. Contribution of various substrates to total citric acid cycle flux and ]anaplerosis as
determined by 13C isotopomer analysis and O2 consumption in the heart.
MAGMA 4: 35–46, 1996.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00407.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
of the citric acid cycle, succinyl-CoA (20, 22, 29, 38). In the
current study, anaplerosis was stimulated by propionate, as
reported previously (Table 1). The role of cardiac anaplerosis
in health and disease is only partially understood, but it
remains a potential target for therapy. Propionate itself has
been suggested as a compound useful for replenishing depleted
pools of TCA cycle intermediates (5), and propionyl-L-carnitine has beneficial effects in animal models and human subjects
(6, 13). There are currently no methods for detecting the effects
of propionate on the human heart. Because multislice cardiac
imaging of HP [13C]bicarbonate is feasible in the pig heart (15)
and administration of HP [1-13C]pyruvate is safe in human
patients (28), extension of these results to human cardiac
imaging is feasible.
The cytosol of the heart is considered by many as a homogeneous space where exogenous pyruvate presumably has free
access to high-activity cytosolic enzymes such as lactate dehydrogenase and alanine aminotransferase. Nevertheless, earlier studies (7, 30, 31) found that 14C labeling in alanine and
lactate derived from exogenous [1-14C]pyruvate was not consistent with a single pool of pyruvate freely exchanging with
alanine and lactate. The current results confirm earlier studies
with 14C that alanine enrichment is consistently significantly
higher than lactate (Fig. 1D), and this discrepancy is enhanced
by propionate. Furthermore, as elucidated in the [U-13C]glucose experiment (data not shown), considerable lactate was
produced in these hearts via glycolysis, yet little of that carbon
source was oxidized in the TCA cycle (0 –3%). The appearance
of [13C]bicarbonate generated from [1-13C]pyruvate is not
proportional to glucose oxidation. Glucose and pyruvate,
under certain conditions, can have very different metabolic
fates in the myocardium. Future experiments that vary the
perfusion conditions will test whether this conclusion can be
further generalized in the perfused heart. In general, the HP
13
C signal from any metabolite is proportional to (fractional
enrichment)·(polarization)·(concentration). Because we have
shown here that exogenous [1-13C]pyruvate does not equilibrate with total lactate in heart tissue, the HP [1-13C]lactate
signal must also underestimate total tissue lactate in these
experiments. Meticulous optimization of pulse sequence design has allowed imaging of HP [13C]bicarbonate and [113
C]lactate, but results here suggest that lactate derived from
exchange with exogenous HP pyruvate might underestimate
the size of the lactate pool (36). Furthermore, the high alanine
enrichment does not parallel lactate enrichment. Oftentimes,
the lactate-to-alanine ratio is taken as a surrogate marker of the
redox state of the cytosol. In this perfusion condition at least,
assessing the redox state by making a ratio of the lactate to
alanine signal intensity would lead to fallacious results. This
study stresses the importance of gaining a complete understanding of the effect of the multiple compartments available to
the heart in vivo.
Innovative Methodology
PROPIONATE STIMULATES PYRUVATE OXIDATION
30. Peuhkurinen KJ, Hassinen IE. Pyruvate carboxylation as an anaplerotic
mechanism in the isolated perfused rat heart. Biochem J 202: 67–76, 1982.
31. Peuhkurinen KJ, Hiltunen JK, Hassinen IE. Metabolic compartmentation of pyruvate in the isolated perfused rat heart. Biochem J 210:
193–198, 1983.
32. Randle PJ, Newsholme EA, Garland PB. Regulation of glucose uptake
by muscle. Effects of fatty acids, ketone bodies and pyruvate, and of
alloxan-diabetes and starvation, on the uptake and metabolic fate of
glucose in rat heart and diaphragm muscles. Biochem J 93: 652–665, 1964.
33. Schroeder MA, Atherton HJ, Cochlin LE, Clarke K, Radda GK,
Tyler DJ. The effect of hyperpolarized tracer concentration on myocardial
uptake and metabolism. Magn Reson Med 61: 1007–1014, 2009.
34. Schroeder MA, Atherton HJ, Heather LC, Griffin JL, Clarke K,
Radda GK, Tyler DJ. Determining the in vivo regulation of cardiac
pyruvate dehydrogenase based on label flux from hyperpolarised [113
C]pyruvate. NMR Biomed 24: 980 –987, 2011.
35. Schroeder MA, Cochlin LE, Heather LC, Clarke K, Radda GK, Tyler
DJ. In vivo assessment of pyruvate dehydrogenase flux in the heart using
hyperpolarized carbon-13 magnetic resonance. Proc Natl Acad Sci USA
105: 12051–12056, 2008.
36. Schroeder MA, Lau AZ, Chen AP, Gu Y, Nagendran J, Barry J, Hu
X, Dyck JRB, Tyler DJ, Clarke K, Connelly KA, Wright GA, Cunningham CH. Hyperpolarized 13C magnetic resonance reveals early- and
late-onset changes to in vivo pyruvate metabolism in the failing heart. Eur
J Heart Fail 15: 130 –140, 2013.
37. Sherry AD, Jeffrey FM, Malloy CR. Analytical solutions for 13C
isotopomer analysis of complex metabolic conditions: substrate oxidation,
multiple pyruvate cycles, and gluconeogenesis. Metab Eng 6: 12–24,
2004.
38. Sherry AD, Malloy CR, Roby RE, Rajagopal A, Jeffrey FM. Propionate metabolism in the rat heart by 13C nmr spectroscopy. Biochem J 254:
593–598, 1988.
39. Stowe KA, Burgess SC, Merritt M, Sherry AD, Malloy CR. Storage
and oxidation of long-chain fatty acids in the C57/BL6 mouse heart as
measured by NMR spectroscopy. FEBS Lett 580: 4282–4287, 2006.
40. Sundqvist KE, Heikkila J, Hassinen IE, Hiltunen JK. Role of NADP⫹
(corrected)-linked malic enzymes as regulators of the pool size of tricarboxylic acid-cycle intermediates in the perfused rat heart. Biochem J 243:
853–857, 1987.
41. Sundqvist KE, Peuhkurinen KJ, Kalervo Hiltunen J, Hassinen IE.
Effect of acetate and octanoate on tricarboxylic acid cycle metabolite
disposal during propionate oxidation in the perfused rat heart. Biochim
Biophys Acta 801: 429 –436, 1984.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00407.2014 • www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on May 4, 2017
18. Malloy CR, Merritt ME, Dean Sherry A. Could 13C MRI assist clinical
decision-making for patients with heart disease? NMR Biomed 24: 973–
979, 2011.
19. Malloy CR, Sherry AD, Jeffrey FM. Analysis of tricarboxylic acid cycle
of the heart using 13C isotope isomers. Am J Physiol Heart Circ Physiol
259: H987–H995, 1990.
20. Malloy CR, Sherry AD, Jeffrey FMH. Evaluation of carbon flux and
substrate selection through alternate pathways involving the citric acid
cycle of the heart by 13C NMR spectroscopy. J Biol Chem 263: 6964 –
6971, 1988.
21. Malloy CR, Thompson JR, Jeffrey FM, Sherry AD. Contribution of
exogenous substrates to acetyl coenzyme A: measurement by 13C NMR
under non-steady-state conditions. Biochemistry 29: 6756 –6761, 1990.
22. Martini WZ, Stanley WC, Huang H, Rosiers CD, Hoppel CL, Brunengraber H. Quantitative assessment of anaplerosis from propionate in pig
heart in vivo. Am J Physiol Endocrinol Metab 284: E351–E356, 2003.
23. Mayer D, Yen YF, Josan S, Park JM, Pfefferbaum A, Hurd RE,
Spielman DM. Application of hyperpolarized [1-13C]lactate for the in
vivo investigation of cardiac metabolism. NMR Biomed 25: 1119 –1124,
2012.
24. Merritt ME, Harrison C, Sherry AD, Malloy CR, Burgess SC. Flux
through hepatic pyruvate carboxylase and phosphoenolpyruvate carboxykinase detected by hyperpolarized 13C magnetic resonance. Proc Natl
Acad Sci USA 108: 19084 –19089, 2011.
25. Merritt ME, Harrison C, Storey C, Jeffrey FM, Sherry AD, Malloy
CR. Hyperpolarized 13C allows a direct measure of flux through a single
enzyme-catalyzed step by NMR. Proc Natl Acad Sci USA 104: 19773–
19777, 2007.
26. Merritt ME, Harrison C, Storey CJ, Sherry AD, Malloy CR. Inhibition
of carbohydrate oxidation during the first minute of reperfusion after brief
ischemia: NMR detection of hyperpolarized 13CO2 and H13CO3⫺. Magn
Reson Med 60: 1029 –1036, 2008.
27. Moreno KX, Sabelhaus SM, Merritt ME, Sherry AD, Malloy CR.
Competition of pyruvate with physiological substrates for oxidation by the
heart: implications for studies with hyperpolarized [1-13C]pyruvate. Am J
Physiol Heart Circ Physiol 298: H1556 –H1564, 2010.
28. Nelson SJ, Kurhanewicz J, Vigneron DB, Larson PE, Harzstark AL,
Ferrone M, van Criekinge M, Chang JW, Bok R, Park I, Reed G,
Carvajal L, Small EJ, Munster P, Weinberg VK, Ardenkjaer-Larsen
JH, Chen AP, Hurd RE, Odegardstuen LI, Robb FJ, Tropp J, Murray
JA. Metabolic imaging of patients with prostate cancer using hyperpolarized [1-13C]pyruvate. Sci Transl Med 5: 198ra108, 2013.
29. Nuutinen EM, Peuhkurinen KJ, Pietilainen EP, Hiltunen JK, Hassinen IE. Elimination and replenishment of tricarboxylic acid-cycle intermediates in myocardium. Biochem J 194: 867–875, 1981.
H1141