IJMS - Free Full-Text - Mitochondrial Transport in Glycolysis and Gluconeogenesis - Achievements and Perspectives

21/02/2024, 09:22 IJMS | Free Full-Text | Mitochondrial Transport in Glycolysis and Gluconeogenesis: Achievements and Perspectives
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lens
Abstract
lens
Introduction
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How Mitochondrial Traffic in
Energy Metabolism Is Easily and
Quickly Investigated?
lens
Cytosolic NADH Oxidation via
the Mitochondrial Shuttles
lens
Phosphoenolpyruvate (PEP)
Transport in Mitochondria
lens
L-Lactate Transport in
Mitochondria
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Author Contributions
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Funding
lens
Institutional Review Board
Statement
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Informed Consent Statement
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Data Availability Statement
lens
Acknowledgments
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Conflicts of Interest
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References
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1 Department of Biomedical Sciences and Human Oncology, University of Bari “Aldo Moro”,
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2 Department of Anesthesiology and Perioperative Medicine, School of Medicine, University
of Louisville, Louisville, KY 40202, USA
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3 Clinica Medica “A. Murri”, Department of Biomedical Sciences and Human Oncology,
University of Bari “Aldo Moro”, 70124 Bari, Italy
* Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(23), 12620; https://doi.org/10.3390/ijms222312620

(https://doi.org/10.3390/ijms222312620)
Submission received: 29 October 2021 / Revised: 16 November 2021 /

Accepted: 19 November 2021 / Published: 23 November 2021
(This article belongs to the Special Issue Mitochondrial Transport and Energy Metabolism
in Health and Diseases ( /journal/ijms/special_issues/mitochondrial_transport ))
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Abstract
Some metabolic pathways involve two different cell components, for instance, cytosol and
mitochondria, with metabolites traffic occurring from cytosol to mitochondria and vice versa,
as seen in both glycolysis and gluconeogenesis. However, the knowledge on the role of
mitochondrial transport within these two glucose metabolic pathways remains poorly
understood, due to controversial information available in published literature. In what follows,
we discuss achievements, knowledge gaps, and perspectives on the role of mitochondrial
transport in glycolysis and gluconeogenesis. We firstly describe the experimental approaches
for quick and easy investigation of mitochondrial transport, with respect to cell metabolic
diversity. In addition, we depict the mitochondrial shuttles by which NADH formed in glycolysis
is oxidized, the mitochondrial transport of phosphoenolpyruvate in the light of the occurrence
of the mitochondrial pyruvate kinase, and the mitochondrial transport and metabolism of L-
lactate due to the L-lactate translocators and to the mitochondrial L-lactate dehydrogenase
located in the inner mitochondrial compartment.
Keywords: mitochondrial transport (/search?q=mitochondrial+transport); glycolysis
(/search?q=glycolysis); gluconeogenesis (/search?q=gluconeogenesis); mitochondrial
shuttles (/search?q=mitochondrial+shuttles); phosphoenolpyruvate (/search?
q=phosphoenolpyruvate); L-lactate (/search?q=L-lactate)
1. Introduction
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Mitochondria play a key role in cell metabolism, and they govern a significant cross talk
with the cytosol. This task is achieved by sharing metabolic
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pathways which rely on both
cytosolic and mitochondrial enzymes. Mitochondria export metabolites/ATP for cytosol
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anaplerosis and import metabolite/ADP for final oxidation and ATP synthesis. Given that
mitochondria are “close spaces” within the cytosol, they possess several translocators
involved in cytosol communication with the inner mitochondrial compartments. However, as
reported by Taylor [1] “the transport selectivities of many carriers remain unknown, and most
have not been functionally investigated in mammalian cells”. A review described “the
multifaced contributions of mitochondria to cellular metabolism” but the transport and
metabolism of certain metabolites in mitochondria, including L- and D-lactate and glutamine,
 
were ignored or incompletely reported [2]. Papers describing the role of mitochondria in
cancer do not take into consideration key transport processes, e.g., L-lactate which is the
main product of cancer cell energy metabolism [3,4,5,6,7,8].
In what follows we describe the metabolite trafficking occurring across the mitochondrial
inner membrane. We focus on aspects of energy metabolism, glycolysis, and
gluconeogenesis and provide answers to questions not yet addressed or without a definite
conclusion. This review is dedicated to biochemists working on novel transport processes
reported in the last two decades, as well as other pathways not included in previous reviews
[9,10,11,12]. Certain issues are revisited considering new experimental findings pointing to
new models/hypotheses. An important point is that each cell has its own metabolism with a
specific role for mitochondria. Thus, it is impossible to consider transport features of each
substrate as common for mitochondria isolated from different sources. In this respect, yeast
mitochondria cannot represent the ultimate model of transport and metabolism of both
mammalian and plant mitochondria. Using simple and rapid methods provides better insights
into the role of mitochondrial transport in the energy metabolism of a specific cell type. Both
isolated, coupled mammalian mitochondria and cell/cell homogenates containing coupled
mitochondria should be considered as ideal models for metabolite traffic studies. In this
review we will address the following questions:
How mitochondrial traffic in energy metabolism can be quickly and easily investigated in
vitro with intact, coupled mitochondria/cell homogenates containing intact, coupled
mitochondria?
How cytosolic reducing equivalents, formed as NADH in glycolysis, are oxidized by
mitochondria?
Anything new about PEP transport in mitochondria in the light of the existence of
pyruvate kinase (PK) in the mitochondrial matrix?
Anything new about L-lactate mitochondrial transport and metabolism in neuronal and
cancer mitochondria? Has this L-lactate-mitochondria affair reached a final conclusion?
2. How Mitochondrial Traffic in Energy Metabolism Is Easily and Quickly Investigated?

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The investigation of the role of mitochondrial transport in energy metabolism relies on the
following aspects:
i. The nature of the involved metabolites.
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ii. The concentration of metabolites in experiments (likely using physiological concentrations).
ii. If and how the nature of the transport and concentration of metabolites change over time
due to variations in enzymatic reactions.
v. Which factors play a role in determining the specificity of a given energy metabolic
pathway.
Importantly, mitochondria should be coupled and intact.
 We essentially, advocate one main type of experimental approach to investigate the
carrier-mediated transport in isolated mitochondria and the intramitochondrial metabolism,
i.e., a “dynamic” approach investigating the kinetics of both transport and metabolism.
Importantly, the closer the conditions of the experiments are to the physiological ones, the
more acceptable the conclusions drawn from the experiments will be. A metabolic flux
depends on its rate-limiting step, and this step must be preferentially identified and
investigated. In the dynamic approach, any parameter can be plotted linearly and correlated
with the concentration as a function of time, while the rate of the reaction is measured as a
tangent of the progress curve. In the dynamic approach, a variety of processes can be
investigated even in the absence of complete identification of the cell components involved.
In a “static” approach, experiments are performed without monitoring the metabolic flux
(see Table 1). Measurements are made afterwards following inactivation procedures of the
investigated molecules. These experiments include genetic, immunological analysis, confocal
microscopy, magnetic resonance spectroscopy, and all the techniques in which mitochondria
are incapacitated as the cell energy power. The approach provides a detailed description of
the molecules involved in transport and metabolism, but no/minimal information on how
certain transports and reactions can play a role in the metabolic pathways.
Table 1. Static approach to investigate the role of mitochondrial transport in energy

metabolism. Pro vs. Contra.
This section summarizes several “older, quick” methods, aimed to encourage further
studies of transport aspects that deal with metabolites and/or mitochondria not yet
investigated. Borrowing from the world of music, these “older” methods could be compared to
Stradivarius or Guarneri violins, i.e., preferable over the modern instruments. Thus, we prefer
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and recommend to researchers interested in transport and metabolism to resort first to simple
and rapid techniques that allow for continuous measurements of metabolite-dependent
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processes with isolated intact, coupled mitochondria or cell homogenates containing intact, searchmenu
coupled mitochondria.
2.1. Spectroscopic Techniques
These techniques include mainly spectrophotometry and fluorescence spectrometry [13].
Spectroscopic methods investigate mitochondrial transport and metabolism. Photometric
measurements can quickly ascertain mitochondrial integrity. Notably, mitochondria can
undergo changes within 1–2 h depending on the experimental conditions (e.g., temperature
and pH). These changes make the comparison difficult in the same experiment since
 
mitochondrial integrity and coupling degrees are different. Adding NADH to the mitochondria
as a test of intactness is strongly recommended, as the absorbance contribution to the
photometer reading is negligible. A constant slow decrease in absorbance at 334/340 nm (the
peak of the reduced nicotinamide) indicates that NADH cannot, or can only poorly be oxidized
by Complex I, located in the inner mitochondrial compartment. Thus, control of mitochondrial
integrity can be established in a very short period and any minor contribution to NADH
decrease can be considered. Evidence of the mitochondrial coupling can be obtained by
checking the capability of an uncoupler to oxidize the intramitochondrial NAD(P)H through its
fluorescence monitoring at 334 and 456 nm excitation and emission wavelength respectively.
Having ascertained both intactness and coupling of the investigated mitochondria, specific
procedures can be then used to study the mitochondrial transport related to intramitochondrial
metabolism.
2.1.1. Spectrophotometry
In agreement with the carrier history, an initial investigation in carrier-mediated
mitochondrial transport could derive by checking certain features of the swelling of
mitochondria suspended in isotonic solutions of a penetrant anion (usually ammonium salt
solutions). Since NH3 can permeate freely the mitochondrial membrane, the occurrence of
mitochondrial swelling in ammonium solutions shows that the investigated anion can enter
mitochondria in a proton compensated manner. Swelling is commonly monitored as a fast
decrease in absorbance, usually at 546 nm, of the mitochondrial suspension that occurs
osmotically with the increase in matrix solute concentration caused by mitochondrial water
uptake. To show that mitochondria are intact (no swelling in sucrose isosmotic solution) and
to gain a first insight into their permeability to metabolites requires less than half an hour and
very small aliquots of mitochondria (about 0.1 mg protein). Following this experiment, we can
answer the following question: is the investigated metabolite penetrant into mitochondria?
The occurrence of swelling, however, does not indicate per se carrier-mediated transport,
since metabolite uptake can also occur via diffusion. Nevertheless, findings such as a stereo-
specific swelling, the inhibition of swelling by a non-penetrant compound, and the swelling
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induction by catalytic concentration of a specific metabolite, strongly indicate a carrier-
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mediated transport. A series of typical swelling experiments are shown in Figure 1. To
ascertain whether and how the transport is energy dependent, the researcher can use certain searchmenu
ionophores under conditions designed to selectively affect ΔpH and ΔΨ. The presence of
nigericin (NIG) or valinomycin (VAL), without added K+ ions, can collapse the mitochondrial
ΔpH which can be increased by adding mitochondria with VAL in the presence of K+ ions.
When swelling is not dependent on the presence of agents allowing dissipation of either ΔpH
or ΔΨ (or both), the penetrant species must have no net charge, as for instance neutral
amino acids.
 
Figure 1. Mitochondrial swelling in transport studies, from Passarella et al. [9]. With
permission from Elsevier, 2021.
The existence of antiport processes is suggested by the swelling occurring only as a

result of the addition of a catalytic amount of one or more counter-anion/s (INDUCER) to
mitochondria suspended in ammonium metabolite solutions.
For instance, the different degrees of swelling of cerebellar granule cell mitochondria
shown for L- and D-lactate (L-LAC and D-LAC) ammonium solutions [14] clearly indicate that
the lactate isomer/s can enter mitochondria in different ways. Given that this cannot depend
on diffusion, due to the similar structure, the existence of one or two different carrier-mediated
transport can be deduced. Swelling inhibition due to a non-penetrant compound by itself

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indicates the occurrence of a carrier-mediated transport. Swelling studies are qualitative and
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of limited interest since they cannot give any indication about the rate of the transport and the
amount of the metabolite taken up by mitochondria or of a substrate affinity to its carrier; searchmenu
nonetheless, investigation of metabolite transport using swelling measurements combined
with stereo-specificity and inhibition studies is strongly recommended. This approach
provides the first insight into its mitochondrial permeability in any cell.
2.1.2. Spectrofluorimetry
Most substrates taken up by mitochondria, and products formed from an imported
metabolite are oxidized in the mitochondrial matrix. The transport of such substrates can be
monitored photometrically (with the pioneering double-beam double wavelength photometer)
 
and fluorimetrically, via changes in the intramitochondrial cofactor red/ox state (Figure 2).
Chappell and Haarhoff [15] first reported ox/red changes in the intramitochondrial pyridine
nucleotides due to the uptake and metabolism of substrates catabolized by pyridine
dehydrogenases. Figure 2A shows such a change where the transport of an oxidable
compound is studied upon fluorescence increase, (λex: 334 nm; λem: 456 nm) resulting from
an intramitochondrial pyridine nucleotide reduction.
Figure (/)
2. Intramitochondrial cofactor red/ox changes in transport studies. Depending on
the context, fluorescence changes are studied as increase (A) or decrease (B,C). For
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details see the text. Abbreviations: AA, antimycin; CN, cyanide; FCCP, carbonyl cyanide
p-trifluoromethoxy-phenylhydrazone (from Passarella et al. [9]). With permission from
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Elsevier, 2021.
Here, mitochondria are incubated with an uncoupler (for instance carbonyl cyanide p-
trifluoro-methoxy-phenylhydrazone, FCCP), to oxidize the intramitochondrial NAD(P)H; upon
attaining a constant fluorescence value rotenone is added to prevent the newly formed
NAD(P)H from being oxidized by the mitochondrial complex I. An increase in fluorescence of
the intramitochondrial pyridine nucleotide, if it occurs because of substrate addition outside
the
 mitochondria, indicates that this substrate can enter the mitochondrial matrix where either

its specific dehydrogenase or the dehydrogenase of a metabolite newly synthesized in the
mitochondria are located. Obviously, controls are needed that no reaction of the investigated
metabolite can occur outside mitochondria. An increase in fluorescence applies to malate
(MAL), phosphoenolpyruvate (PEP), pyruvate (PYR), and L-lactate (L-LAC) in glycolysis. On
the other hand, as first shown by Haslam and Krebs [16] the uptake of oxaloacetate (OAA),
metabolized by malate dehydrogenase (MDH) in the mitochondria, is measured by the
fluorescence decrease (λex: 334 nm; λem: 456 nm). This is due to the intramitochondrial
pyridine nucleotide oxidation, which occurs upon OAA addition to mitochondria pre-incubated
with rotenone to block NADH oxidation via complex I (Figure 2B). The use of fluorimetric
techniques is appropriate in the dynamic study of transport, and we agree with Mayevsky and
Rogatsky [17] who state that “The large numbers of publications by different groups testify to
the valuable information gathered in various experimental conditions. The monitoring of
NADH levels in the tissue provides the most important information on the metabolic state of
the mitochondria”.
Surprisingly, such a simple procedure has not been appreciated and accepted in a recent
paper [7] stating that “Unfortunately, some studies which have concluded the existence or
absence of mitochondrial LDH, and implied the physiological relevance thereof, base such
claims on experiments involving the continuous monitoring of added NADH autofluorescence
in isolated mitochondria [14,18,19,20,21,22,23]”. We believe that the authors misinterpreted
the cited papers since NADH was not added to mitochondria. Rather, the authors monitored
only the fluorescence of the mitochondrial NAD(P)H [14,18,19,20,21,22,23].
The first demonstration of the uptake of D-LAC into mitochondria, a substrate that is
metabolized by a flavin-dependent dehydrogenase, dates 2002 [24]. Mitochondria were
added with FCCP and an inhibitor of the respiratory chain complex III and the fluorescence
increase at wavelengths 450 and 520 nm, excitation and emission, respectively, was followed
as the measurement of the substrate uptake (Figure 2C). In distinction with NAD(P)H, which
is by itself fluorescent, a flavin fluorescence depends on the physiological environment due to
its function as a prosthetic group.
As discussed later, the researchers must accurately ascertain that the rate of
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fluorescence changes mirrors the rate of the transport, rather than one of the metabolic
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enzymatic reactions. Thus, the application of the control strength criterion and fractional
inhibition, achieved by using non-penetrant compounds, can reveal whether or not the searchmenu
transport is the rate-limiting step of the investigated process and can also provide an initial
indication to ascertain if specific metabolite traffic depends on a single carrier or not.
According to the control strength criterion, if the reciprocal of the measured rate is plotted as
a function of the non-penetrant inhibitor concentration, the resulting Dixon plot (1/V against
[I]), when extrapolated to zero concentration, provides a measure of transport in the absence
of inhibitor; thus, the coincidence of the experimental point measured in the absence of
inhibitor with the intercept indicates that the rate of inhibited step, i.e., the transport, is
measured.
 The data from the Dixon plot could also be plotted as 1/i against 1/[inhibitor],
where the fractional inhibition, i, is 1 − Vi/Vo and Vi and Vo are the rates of the measured
reactions in the presence or absence of inhibitor, respectively. If y-intercept is unity, this
shows that the inhibitor can prevent the reaction completely, i.e., that the reaction cannot
occur via another carrier insensitive to the inhibitor used. Obviously, control strength can be
also applied to enzymatic reactions by using specific inhibitors.
A significant development of the application of spectroscopic methods to the study of
mitochondrial transport was obtained at the end of the 1970s when a variety of
metabolite/compound detecting systems consisting of enzyme/s and cofactor/s were
developed to selectively detect the appearance outside mitochondria of molecules derived
from the mitochondrial metabolism of taken-up substrates.
Interestingly, in the presence of enzymes and cofactors, the reconstruction can be made
of certain metabolic pathways in which cytosol and mitochondria are involved (for refs. see
[9,14]).
Since these detecting system components are mostly cytosolic, and since mitochondrial
metabolism is, at least partially still occurring when they are being used, we consider this
approach to be close enough to the physiological situation.
Figure 3 shows the ATP and OAA detecting systems. ATP and OAA appearance occur
because of the addition of ADP and MAL, respectively, and is shown as absorbance (at 334
nm) increase and decrease, respectively. With fluorimetry (e.g., when measuring the
appearance of ATP outside mitochondria, as a result of ADP addition), the transport can be
studied with mitochondrial protein as low as 0.1 mg, see for instance [25].
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Figure 3. The ATP and the OAA detecting systems. The externally added components
 of the ATP and OAA detecting systems are reported in green. Main abbreviations: ADK, 
adenylate kinase; Ap5A, P1,P5-di(adenosine-5′)penta-phosphate; G6PDH, glucose-6-
phopshate dehydrogenase; 6-P-GLU-d-LAC, 6-phosphoglucono-d-lactone; HK,
hexokinase. From Passarella et al. [9]. With permission from Elsevier, 2021.
When using a metabolite/compound detecting system the reactions occurring outside

mitochondrial must not be the rate-limiting step of the whole process. Interestingly, given that
ATP can be synthesized by mitochondria via both adenylate kinase and ATP synthase, the
use of inhibitors that can selectively inhibit either adenylate kinase (Ap5A) or ATP synthase
(oligomycin) can distinguish the mechanism of ATP production. Importantly, control strength
application with oligomycin as an inhibitor provides a measurement of the ATP synthase
reaction in intact mitochondria even if the rate-limiting step of the monitored ATP efflux is the
rate of the ADP/ATP exchange. In this case, ATP synthase kinetics cannot be followed
directly, however, if the rate of ATP appearance outside mitochondria due to externally added
ADP is measured in the presence of oligomycin. Then the inhibition kinetics can provide
information on the ATP synthesis rate: the intercept at the Y-axis of the linear regression of
rate values obtained for oligomycin concentrations that produce inhibition of the rate of
fluorescence increase represents the reciprocal of the rate of non-inhibited ADP
phosphorylation via ATP synthase. By plotting the intercept values as a function of reciprocal
ADP concentration, a double reciprocal plot can be obtained, which provides Vmax and Km
values for ADP in the reaction catalyzed by ATP synthase [26].
Since two or more carriers can contribute to the appearance of a metabolite outside the
mitochondria, specific inhibitors can be used to elucidate the mechanism by which said
metabolite movement occurs. Atlante et al. [27] firstly studied the fumarate (FUM) uptake by
mitochondria using protein detecting systems to monitor the appearance of a mitochondrial
enzyme exported outside mitochondria. Passarella et al. [28] found that the uptake of
aspartate aminotransferase into mitochondria in vitro causes efflux of MDH and vice versa.
Other simple dynamic methods that can be used to show a metabolite mitochondrial transport
and metabolism include measurements of oxygen uptake, pH changes in the
extramitochondrial phase, and measurements of mitochondrial ΔΨ generation. Having

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established that the transport is the rate-limiting step of the monitored process, studies have
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shown that L-LAC uptake occurs into a variety of mitochondria, as normal prostate cells [10],
cancer prostate cells [20], Hep G2 cells [21], and rabbit gastrocnemius mitochondria [22]. The searchmenu
different pH profiles and the different sensitivity to non-penetrant inhibitors, together with
measurements of the Vmax in kinetic experiments carried out under the same experimental
conditions, allow for the identification of the different carriers. For instance, evidence was
found that rat liver mitochondria (RLM) possess the carriers that mediate L-LAC/H+ symport,
L-LAC/OAA and L-LAC/PYR antiporters which proved to differ from one another and with
respect to the D-LAC/H+ and PYR/H+ symporters and the D-LAC/malate and D-LAC/oxoacid
antiporters [19] and that the different carriers transport glutamine in normal and acidotic rat
kidney
 mitochondria (RKM) (see [9]). 
2.2. Isotopic Techniques
Isotopic techniques directly study the transport, besides determining the chemical
modification of a substrate into a metabolic product in a biological sample. The techniques
appeared in the seventies of the last century to confirm the occurrence of metabolite
transport. Direct methods for measuring metabolite transport and distribution in mitochondria
have been reported in detail in the paper of Palmieri et al. [29], and here we discuss a
simplified method to investigate transport kinetics, both in net uniport/symport-dependent
radioactivity uptake and antiport-dependent exchanges between externally added and
labelled substrates already present upon loading in mitochondria. In isotopic kinetic
investigations of transport, mitochondrial metabolism must be blocked. Such blockade
appears to be particularly useful since it allows for measuring only the substrate via uptake; in
this case, the labeled substrate uptake is blocked using a non-penetrant inhibitor in a time
range of seconds. The amount taken up is calculated by subtracting the radioactivity
measured in mitochondria when the substrate is added in the presence of the inhibitor from
the radioactivity measured in the organelles when the inhibitor is added a few seconds after
the labeled substrate addition.
In a typical experiment, mitochondria (about 50 μL that contain 1–2 mg protein) are
suspended in a standard medium in an Eppendorf cup containing tritium-labeled water
(3H2O) added for measuring the pellet volume. Mitochondria (usually kept in a vial put in ice +
water to prevent temperature-dependent uncoupling) are incubated for approximately 1 min to
allow equilibration at the experimental chosen temperature. Next, the following are added
rapidly:
A. The 14C-labelled substrate followed few seconds later (1–20, depending on the time when
the transport is proved to be linear) by the inhibitor/s (at a concentration that should result
in total inhibition) or
B. First the inhibitor/s followed by the labelled substrate.
Several seconds after these additions, the cup is placed in a microcentrifuge for 1 min to
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form a supernatant and a pellet (mitochondria). The supernatant is completely removed and
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saved, and the pellet is suspended first in 100 μL water, followed by 50 μL of 7 N perchloric
acid and vigorous shaking. This treatment dissolves the mitochondria. Upon additional searchmenu
centrifugation, the mitochondrial content should be in the supernatant. Notice that the pellet
and the obtained supernatant contain both 14C and 3H radioactivity. The 14C radioactivity of
the taken-up substrate (ni) in nmol is deduced from the total radioactivity in nmol (ntot) minus
the radioactivity in nmol attached to the mitochondrial outer membrane and/or the label
remained in the extramitochondrial space (ne). Since H2O can enter mitochondria, the total
3H radioactivity is the sum of that included in the mitochondria and that present outside the
mitochondria.
 A procedure must be followed by which the nmol taken up by mitochondria can be 
calculated.
The nmol of labeled substrate found in the pellet ntot are equal to ni + ne and therefore:
ni = ntot − ne
ntot = 14Ccpm/SA
where 14Ccpm is the counts/min of each sample, as measured with a radioactivity counter
and SA is the specific activity measured as 14Ccpm/nmol. This value can be calculated by
measuring the radioactivity of a sample in which a known number of nmol of labeled substrate
is added.
Therefore:
ni = 14Ccpm/SA − 3Hcpm/SAH2O × Ce − ViCe
Thus, to calculate ni, ne must be measured.

The nmol taken up by mitochondria are essentially negligible with respect to those
present in the initial suspension at the substrate concentration (Ce). (For instance, if the
substrate concentration Ce added in the Eppendorf cup is 1 mM or 1000 nmol/mL and since
usually the taken-up substrate in nmol is <5 nmol, Ce can be considered unchanged).
Therefore, ne can be measured as the product of Ve × Ce where Ve is the volume of water
outside mitochondria. Obviously, Ve = Vtot − Vi where Vtot is the volume of the pellet including
3H O inside mitochondria and the volume of 3H O outside mitochondria and V is the volume
2 2 i
of water inside mitochondria.
Thus:
ne = (Vtot − Vi) × Ce; ne = Vtot × Ce − Vi × Ce
Vtot can be obtained by measuring the radioactivity of 3H2O/SAH2O where SAH2O is the
specific activity of the labeled water expressed as cpm of 3H2O/μL which can be measured
from the cpm of 3H2O added in 1 mL of the medium in the Eppendorf cup.
Therefore:
ni = 14Ccpm/SA − 3Hcpm/SAH2O × Ce − ViCe
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As a reminder, while the experiment described above is carried out under:
Condition A: Substrate → Inhibitor
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in a parallel experiment, carried out simultaneously, the inhibitor is added before the searchmenu
labeled substrate such that the radioactivity remained outside of the mitochondria (perhaps
with some label bound to the outer membrane)
Condition B: Inhibitor → Substrates
Hence, the taken-up substrate in nmol can be calculated as the difference between
condition A and condition B measurements:
ni = 14Ccpm/SA − 3Hcpm/SAH2O × Ce − ViCe (under A condition) −
ni = 14Ccpm/SA − 3Hcpm/SAH2O × Ce − ViCe (under B condition).

 
Importantly, Vi × Ce, which is constant under both conditions, will be eliminated.
Notably, fluctuations that may result from the use of separate mitochondrial preparations
can be eliminated when an experiment runs using the same preparation to include
simultaneous measurements under both conditions A and B. Moreover, increasing the
number of samples (3 or more) should allow the results to be expressed as mean +/−
standard error.
Measurements can be made of the radioactivity of the 14C-substrate loaded mitochondria
to monitor the exchange between an externally added metabolite/compound and the labeled
metabolite/compound present in mitochondria after loading. In this case, the exported
mitochondrial labeled metabolite/compound will be obtained as the difference of the
radioactivity measured under condition B—the radioactivity measured under condition A.
Regrettably, isotopic techniques cannot distinguish between compounds that are taken
up and similar compounds that are newly metabolized intramitochondrially from the portion of
the taken-up substrate that is insensitive to penetrant inhibitors. Thus, the lack of a significant
radioactivity accumulation in mitochondria due to the antiport between two labelled
compounds could obscure the existence of several transport processes. For instance, in
isotopic experiments, FUM was considered to be a non-penetrant anion due to an antiport
that occurs between externally added FUM and malate, such that newly synthesized malate
intramitochondrially from taken up FUM was exported to the extramitochondrial phase (see
ref. [9]). Although the use of labeled substrates was fundamental in discovering and
investigating in some detail metabolite transport, the necessity to impair mitochondrial
metabolism makes mitochondria just a passive recipient of the incoming substrate without
any ability to control or modulate intramitochondrial metabolism.
Independently of the techniques used when studying transport via a dynamic approach,
since carrier proteins behave as enzymes in catalyzing metabolite movement across the
mitochondrial membrane, any carrier-mediated transport could be characterized according to
several criteria: the occurrence of saturation kinetics, the pH and temperature dependence,
substrate specificity, the sensitivity to non-penetrant compound/s, the occurrence of antiport
stoichiometry, the energy dependence, etc. Accordingly, any difference found among the
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investigated features should indicate the existence of separate transport mechanisms and
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consequently, separate carriers. It should be noted that since the traffic of metabolites can be
derived from the combined action of two or more translocators (see below), this point must be searchmenu
taken into consideration when studying physiological metabolite movement.
Notice that Vmax, Km, and Ki values have only relative significance here since they are
dependent on a variety of parameters essentially linked to medium composition (buffer, ionic
strength, nature of ions, pH, etc.). However, they can be used in comparative studies.
Along with isotopic techniques, the stop inhibitor method can be applied to any analytical
technique used to measure take up or efflux of substrate including for instance HPLC.
2.3. Final Comments
 
In the last 60 years, the mitochondrial transport has been investigated both via a dynamic
and a static approach. We confirm that to study the role of the mitochondrial carrier in energy
metabolism the experimental approaches described above are preferable.
Finally, we stress that the application of sophisticated techniques to study transport and,
in particular, metabolism appears to be expensive and not simple to be used in the
biochemistry laboratory: for instance, the conclusion that L-LAC addition to cancer cells can
contribute to lipid synthesis proposed by Chen et al. [30] using high-resolution mass
spectroscopy and transmission electron microscopy was a finding already published using
essentially spectroscopic techniques: Chen et al. claim that their findings “demonstrate a link
between lactate metabolism and the mitochondria of fermenting mammalian cell” however
such a proposal was made 4 years before by showing that L-LAC addition to Hep G2 cell
homogenates containing intact and coupled mitochondria resulted in the appearance of
citrate, the precursor of fatty acid synthesis, in the extramitochondrial phase [21].
3. Cytosolic NADH Oxidation via the Mitochondrial Shuttles
In glycolysis, NADH is formed in the reaction catalyzed by the glyceraldehyde-3-

phosphate dehydrogenase. Given that NADH cannot enter mitochondria, its oxidation can
occur both via L-lactate dehydrogenase (L-LDH) in the cytosol, with L-LAC production from
PYR (with 2 ATP formed in substrate-level phosphorylation), and via the mitochondrial
shuttles (with more than 30 ATP formed in the oxidative phosphorylation). A mitochondrial
shuttle is a process in which enzymes and mitochondrial carriers transfer into or out of
mitochondria molecules and/or charges that do not cross the mitochondrial inner membrane
via simple diffusion. Important shuttles transfer reducing equivalents formed in glycolysis,
where NADH is oxidized in the cellular cytosol by specific dehydrogenases and where the
resulting reduced metabolites are then shuttled into the mitochondria. Then, specific
mitochondrial enzymes re-oxidize these metabolites with concomitant reduction in
mitochondrial cofactors (FAD/NAD+). These cofactors are then oxidized in the mitochondrial
respiratory chain as electrons flow to oxygen, generating the electrochemical proton gradient
used in the oxidative phosphorylation process that forms ATP. Finally, either directly, with no
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transport, in the case of the glycerol-3-phosphate/dihydroxy-aceton-phosphate (G3P/DHAP)
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shuttle, or indirectly, in a process that utilizes carrier-mediated transport, oxidized compounds
are returned to the cytosol for the further reaction involved with NADH. The mitochondrial searchmenu
shuttles which allow for cytosolic NADH oxidation have already been reported in [9]. These
are the malate/oxaloacetate (MAL/OAA), the malate/aspartate (MAL/ASP), the
glycerolphosphate/dihydroxyacetone-phosphate (G3P/DHAP), the proline/glutamate
(PRO/GLU) and the L-lactate/pyruvate (L-LAC/PYR). Obviously, the single contribution of
each shuttle, as well as its reversibility, depends on the cell type in which they occur.
MAL/OAA, MAL/ASP, G3P/DHAP, and L-LAC/PYR shuttles are involved in glycolysis. We will
consider essentially the first three described in Figure 4. Demonstration of these shuttles in
vitro
 is relatively simple by measuring photometrically the oxidation of externally added NADH 
to intact coupled mitochondria in the presence of the specific substrate and enzyme. In the
case of the G3P/DHAP shuttle, Dawson and Cooney [31] using RKM showed that “the
mitochondrial preparation oxidized added NADH even in the absence of further additions.
However, this oxidation was strongly inhibited by rotenone, suggesting that damaged
mitochondria were responsible for it. With NADH oxidation thus lowered, first
glycerolphosphate dehydrogenase (G3PDH) and then glycerolphosphate G3P were added.
The addition of the enzyme did not affect the NADH oxidation rate, but the introduction of
glycerol-phosphate brought about an immediate and marked increase in the oxidation rate.
This increase was abolished by cyanide, indicating its dependence on the mitochondrial
respiratory chain”.
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 
Figure 4. αGP, MAL/ASP, and MAL/OAA shuttles. MIM, mitochondrial inner membrane.
Mitochondrial translocators: Pi, phosphate carrier; DIC, dicarboxylate translocator;
OXODIC, oxodicarboxylate translocator; GLU/ASP, glutamate/aspartate translocator.
Enzymes: 1, mitochondrial GPDH; 2 and 3, cytosolic and mitochondrial MDH
respectively; 4 and 5, cytosolic and mitochondrial AAT respectively. From Atlante et al.
[32] and Passarella et al. [12]. See text for details.
The malate/oxaloacetate shuttle (MAL/OAA) shuttle has already been proposed in 1960
by Klingenberg and Bucher [33]. It invoked the mitochondrial export of OAA in exchange for
uptake of malate. Many researchers considered this alternative improbable, as the OAA
concentration calculated from the equilibrium constant of the MDH reaction was very low and
because the mitochondrial membrane was (erroneously) thought at the time to be
impermeable to OAA. Indeed, a first indication of the permeability of mitochondria to OAA was
offered by Haslam and Krebs [16] in 1968, later Gimpel et al. [34] suggested that OAA was
transported in RLM via the dicarboxylate carrier. The in vitro transport of OAA in isolated RLM
was later shown by checking the OAA capability to cause efflux of labeled dicarboxylates from
loaded mitochondria [35,36]. In this case, the millimolar OAA concentration, a very high
concentration compared to the micromolar physiological one, poses some doubts about the
possible occurrence of OAA traffic across the mitochondrial membrane in vivo. Nonetheless,
the use of the dicarboxylate carrier in OAA transport was ruled out since Pi efflux, due to
externally added OAA, considered as an indication of OAA transport via the dicarboxylate
carrier, proved to be lacking when malate was removed by using malic enzyme and NADP+
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outside mitochondria. Surprisingly enough, Darvey [37] proposed that OAA can use the
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dicarboxylate and the Pi carrier to enter mitochondria. In another study, the authors used
micromolar OAA concentrations, such as those measured in the cytosol. The capability of searchmenu
externally added OAA (at concentrations such as those measured in the cytosol) to bring
about malate efflux from rat heart mitochondria was shown, as described above, by using the
malate detecting system [38]. This was the first indication for a possible occurrence of a
MAL/OAA shuttle used to export reducing equivalents from mitochondria. The mechanism of
OAA efflux from mitochondria has been investigated because of its possible role in
transferring cytosolic reducing equivalents into mitochondria. Consequently, reconstruction of
the malate/OAA shuttle was shown made in a variety of mitochondria including those isolated
from
 rat brain [39], rat kidney [40], rat cerebellar granule cells [41] saccharomyces cerevisiae

[42], durum wheat and potato cells [43], and heart left ventricle [32]. For instance, the
MAL/OAA shuttle was reconstructed by using non-synaptosomal rat brain mitochondria
(RBM). The results demonstrated the occurrence of a carrier-mediated transport for OAA,
sensitive to dicarboxylate analogs and mersalyl, and of the two isoenzymes of MDH.
Malate/OAA shuttle rate appears to depend on the activity of the malate/OAA exchange
across the mitochondrial membrane. This shuttle could account for the oxygen uptake by
brain slices still occurring (40% of the control) when the transaminase which plays a major
role in the MAL/ASP was 90% inhibited [39]. Interestingly, Pastore et al. [43] used plant
mitochondria and showed that no NAD(P)H oxidation occurred arising from the MAL/ASP and
the G3P/DHAP shuttles, and that NADH is oxidized in the presence of MAL and MDH,
according to the presence of the MAL/OAA shuttle.
To date, the occurrence of the MAL/OAA shuttle is essentially ignored in all the
biochemistry textbooks despite its in vitro reconstruction can be achieved in a 2-min
experiment [44]. Unfortunately, no mention of either MAL/OAA or L-lactate/pyruvate (L-
LAC/PYR) shuttles (see below) could be found in a paper dealing with pyridine nucleotide
regulation of cardiac intermediary metabolism [45]. A study of metabolism in cancer cells
metabolism reported that MAL/ASP shuttle “exerts control over NAD+/NADH homeostasis to
maintain the activity of mitochondrial lactate dehydrogenase and to enable aerobic oxidation
of glycolytic L-lactate in mitochondria”. However, again either MAL/OAA or LAC/PYR shuttles
are not taken into consideration [46]. Again, the MAL/OAA shuttle, the malate cycle in terms
of Klingenberg and Buecher [33], is not considered in a recent review by Borst [47], who
commented about the malate cycle, but ignored all the findings described in other papers
[9,44].
Which is the contribution of these three shuttles in any given type of mitochondria?
Atlante et al. [32] described in detail the role played by the MAL/OAA shuttle in oxidizing
external NADH (with comparison made with to the MAL/ASP and G3P/DHAP shuttles) in left
heart ventricle mitochondria (RHLVM). This issue was addressed by reconstructing
appropriate mitochondrial shuttles in vitro; here, use was made of RHLVM of both
normotensive (WKY) and spontaneously hypertensive (SHR) rats at 5 and 24 weeks of age;
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they were used as model systems for left ventricle normotrophy and
hypertrophy/hypertension, respectively.
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To monitor the activity of the different shuttles, a comparison was made of NADH searchmenu
oxidation between mitochondria isolated from SHR and WKY rats.
How the three investigated shuttles have been reconstructed in vitro is shown in some
detail in Figure 5. In all cases, mitochondria were initially incubated with NADH (0.2 mM) and
the absorbance at 340 nm was monitored for a short period of time; the constancy of
absorbance at 340 nm demonstrated that the internal mitochondrial Complex I was
inaccessible to NADH, i.e., the intactness of mitochondria. Then, the appropriate substrates
and enzymes were added to reconstruct either the α-GP or the MAL/ASP shuttles and to
establish
 the occurrence of the malate/OAA shuttle. 
Figure 5. Oxidation of extramitochondrial NADH by WKY5-RHLVM. Reconstruction of

the aGP shuttle (A), of the overstimated malate/aspartate (MAL/ASP) shuttle (B), of the
malate/oxaloacetate (MAL/OAA) (C), of the true MAL/ASP shuttle (D). From [12,32],
with permission from Arcane Editrice, Rome and Spandidos Publications, 2021.
Unloaded (A, B(a) and C) or mitochondria loaded with aminooxyacetate (AOA), an

amino-transferase inhibitor which can enter mitochondria were incubated at 25 °C in 2.0 mL
of standard medium, consisting of 0.2 M sucrose, 10 mM KCl, 20 mM HEPES-Tris, pH 7.2
and 1 mM MgCl2, plus 0.2 mM NADH in the presence of α-glycerolphosphate dehydrogenase
(α-GPDH, 0.5 e.u.) (A), malate dehydrogenase (MDH, 2 e.u.) (C), α-oxoglutarate (0.1 mM)
plus MDH plus aspartate aminotransferase (AAT, 1 e.u.) (B and D). Where indicated,
additions were as follows: 1 mM α-glycerolphosphate (α-GP), 1 mM glutamate (GLU), 1 mM
malate (MAL), 10 mM phenylsuccinate, (PHESUCC) a non-penetrant compound known to
inhibit both the dicarboxylate and the oxodicarboxylate carriers. Confirmation was made that
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in AOA-loaded mitochondria AAT was strongly inhibited (B, inset); in fact, RHLVM, unloaded
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(C) or AOA-loaded mitochondria, suspended in 2 mL of standard medium in the presence of 2
µg rotenone and 1 mM sodium arsenite, were added with aspartate (ASP, 12 mM) and, after 1 searchmenu
min, with α-oxoglutarate (α-OG, 3 mM). The rate of decrease in fluorescence (λex = 334 nm/
λem = 456 nm) was then recorded and taken as a measurement of intramitochondrial AAT
activity. Numbers along curves are rates of change in absorbance at 340 nm measured as
tangents to the initial part of the progress curves and expressed as nmol NADH oxidized/min
× mg mitochondrial protein.
In all cases, the operation of the shuttle was monitored continuously as a decrease with
time of the NADH absorbance at 340 nm arising from metabolite interaction with the
mitochondrial
 α−glycerol-phosphate dehydrogenase (m-GPDH), in the case of αGP/DHAP 
shuttle, or from traffic across the mitochondrial membrane (in the case of the other two
shuttles) as revealed using specific substrate detecting systems (see Table 2). In Figure 5A,
1 e.u. α-GPDH was added to mitochondria. The DHAP concentration in the
extramitochondrial phase was negligible since no change in absorbance occurred. As a result
of the addition of 1 mM α-GP, a decrease in absorbance was observed at a rate equal to 4.6
nmol NADH oxidized/min × mg mitochondrial protein. The explanation for this finding (Figure
5A) is that added α-GP is oxidized by mitochondria in the reaction catalyzed by mitochondrial
α-glycerolphosphate dehydrogenase (m-GPDH) located in the outer phase of the inner
mitochondrial membrane and the product DHAP, in turn, oxidizes NADH outside the
mitochondria in the reaction catalyzed by the added α-GPDH. Initial attempts to measure
NADH oxidation as a result of the operation of the MAL/ASP shuttle were carried out as
follows: mitochondria were incubated with the aspartate detecting system (ASP D.S.),
consisting of 0.2 mM NADH, 0.1 mM α−ketoglutarate (α-KG), 0.5 e.u. malate dehydrogenase
(MDH) plus 1 e.u. aspartate aminotransferase (AAT). Then, malate plus glutamate (1 mM
each) were added to the sample and the change in absorbance was measured (Figure 5B).
In this case, efflux of aspartate from the mitochondria was followed by its transamination via
AAT to OAA, which in turn is reduced to malate in a reaction catalyzed by MDH outside
mitochondria. The rate of decrease in absorbance was equivalent to 11 nmol NADH
oxidized/min × mg mitochondrial protein (Figure 5B(a)). It must be emphasized that this is a
considerable overestimate of the true activity of the shuttle as demonstrated by experiments
using aminooxyacetate (AOA), (an AAT inhibitor which can enter mitochondria): first, it was
confirmed that loading of mitochondria with AOA resulted in, essentially, a complete inhibition
of mitochondrial AAT activity (Figure 5B, inset): when aspartate and α-ketoglutarate, the
transaminase substrate pair, were added to “control” mitochondria a fast decrease in the
fluorescence of the intramitochondrial pyridine nucleotides occurred. This decrease occurred
because of OAA formation inside the mitochondria via mitochondrial AAT and its reduction to
malate via MDH.
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Table 2. The compound/metabolite detection outside mitochondria.
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When using mitochondria loaded with AOA, no significant change in fluorescence was
observed, indicating a complete inhibition of mitochondrial AAT. Given the mode of operation
of the MAL/ASP shuttle, the shuttle should not occur in AOA-loaded mitochondria. However,
when AOA-loaded mitochondria were used, the rate of NADH oxidation only decreased by
approximately
 40% to 6.5 nmol NADH oxidized/min × mg mitochondrial protein (Figure 
5B(b)). This residual activity cannot be due to the MAL/ASP shuttle operation, which requires
transamination in the mitochondrial matrix, and the true shuttle activity must be represented
by the difference between the values in Figure 5B(a,b), i.e., 4.5 nmol NADH oxidized/min ×
mg mitochondrial protein (see below). One possible explanation for the residual activity is in
Figure 5B(b) is that an MAL/OAA shuttle exists; this would be consistent with the permeability
of heart mitochondria to OAA [9]. Direct evidence for this hypothesis has been obtained
(Figure 5C): when mitochondria were incubated with MDH, (0.5 e.u.) and NADH alone, i.e.,
the OAA detecting system (OAA D.S.), no change in absorbance occurred showing that the
OAA concentration in the extramitochondrial phase was negligible. Externally added 1 mM
malate (MAL) caused oxidation of NADH at a rate of approximately 6.8 nmol/min × mg
protein, indicative of the appearance of OAA outside the mitochondria. NADH oxidation was
strongly inhibited (~85%) by 10 mM phenylsuccinate. The explanation of these findings is the
following: MAL can enter mitochondria in exchange with endogenous phosphate or
dicarboxylates; once inside the matrix, MAL is oxidized by mitochondrial MDH to OAA, which
in turn can exit in a manner sensitive to phenylsuccinate. Once outside mitochondria, it is
reduced by NADH in the presence of MDH in a reconstructed MAL/OAA shuttle.
Based on these findings, indicating that the activity of the MAL/ASP shuttle, as shown in
Figure 5B(a) and previously by others is overestimated, being dependent on the efflux of
both aspartate, which is converted to OAA via transamination, and OAA itself, a new
procedure was developed to continuously monitor MAL/ASP shuttle activity. Here, ASP D.S
was added to the mitochondrial suspension in the sample cuvette, whereas ASP D.S. without
AAT was added to the reference cuvette. Then, malate plus glutamate (1 mM each) were
added simultaneously to both the sample and reference cuvettes and the oxidation of NADH
was recorded. In the sample cuvette (CUV 1) OAA derives from a simple exchange and
because of the aspartate transamination, while in the reference cuvette (CUV 2) only OAA
efflux can be monitored. The absorbance difference between that of CUV 1 and CUV 2 gives
the true rate of MAL/ASP shuttle, which was found to be 4.3 nmol NAD(P)H oxidized/min ×
mg mitochondrial protein (Figure 5D(a)). Consistently, when using AOA-loaded mitochondria,
the activity of the shuttle was effectively abolished (Figure 5D(b)). As expected, the
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measured true activity of the MAL/ASP shuttle was very close to the difference between the
activities shown in Figure 5B(a,b).
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Given that the real contribution of the investigated shuttles depends also on the searchmenu
physiological concentration of the substrates, the dependence of the rate of decrease in
absorbance of NADH, i.e., the rate of the in vitro reconstructed shuttles was investigated as a
function of increasing concentrations of either α-GP or MAL. Hyperbolic reaction kinetics were
found for the α-GP/DHAP MAL/ASP and MAL/OAA shuttles, and Figure 6 shows a typical
double reciprocal plot for data obtained with WKY5-M. Km values, i.e., the substrate
concentration, which gives half the maximum rate (Vmax), and Vmax values were determined
in several experiments carried out with different mitochondrial preparations. The values
obtained
 with SHR5-M, SHR24-M, WKY5-M, and WKY24-M were calculated along with 
statistical analysis using the ANOVA test. In five experiments, the Vmax values of the
MAL/ASP shuttle as measured in SHR5 vs. SHR24 differed significantly both from one
another and from the same-age WHY samples (p < 0.05). In contrast, no statistically
significant differences were found for Vmax values of the α-GP/DHAP shuttle independently
of the age and hypertrophy/hypertension states.
Figure 6. The dependence of the rate of NADH oxidation via the three shuttles on
increasing substrate concentrations. From [12,32], with permission from Arcane Editrice,
Rome and Spandidos Publications, 2021.
Interestingly, in this case, the rate of NADH oxidation was shown to depend on the rate of
MAL/OAA antiport across the mitochondrial membrane. This raises the question as to
whether the metabolite transport processes play a role in metabolic regulation.
In conclusion, most NADH oxidation occurs via the MAL/OAA shuttle, the activity of
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which increases with time and with the progression of hypertrophy and development of
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hypertension as judged by statistical ANOVA analysis. In contrast, the other two shuttles
investigated were shown to make only a minor contribution to NADH oxidation in a manner searchmenu
essentially independent of age and progression of hypertrophy/hypertension. The rate of
NADH oxidation via the MAL/OAA shuttle is the rate of malate transport in exchange with
OAA. Therefore, the contribution of the MAL/OAA shuttle to oxidation of NADH is higher than
those of the MAL/ASP and α-GP shuttles, even if OAA cannot enter mitochondria.
It is somewhat surprising that in various papers dealing with the oxidation of cytosolic
NADH, the occurrence of the MAL/ASP shuttle has been considered without any experiment
carried out to ascertain the OAA efflux. For instance, in a paper by Gregory et al. [48], dealing
with
 reducing equivalent transfer to the mitochondria during gluconeogenesis and 
ureagenesis, the MAL/OAA shuttle was completely ignored. The assumption that OAA cannot
cross the mitochondrial membrane drove the conclusion that “the bulk of oxaloacetate,
formed by PYR carboxylation within the mitochondria during gluconeogenesis from lactate, is
transported to cytoplasm as aspartate” [49].
In a paper aimed to investigate the influence of thyroid hormone on the NADH shuttles in
cardiac and liver mitochondria, MAL/ASP and α-GP/DHAP shuttle capacities were
significantly increased in cardiac mitochondria from adult rats treated for 9 days with T3
compared to saline-treated controls, but a possible role for the MAL/OAA shuttle was ignored
[50]. In another paper by the same group, it was suggested that “sufficient malate/aspartate
and α-glycerophosphate shuttle capacity exists in cardiac mitochondria to accommodate
increased shuttle flux as hypertrophied myocardium becomes more glycolytically active” [51].
We believe that any study of the MAL/ASP shuttle should be accompanied by
experiments in which the occurrence of the MAL/OAA shuttle is also investigated. For
instance, it should be noted that in a study where MAL/ASP shuttle was investigated in
vascular smooth muscle [52] in the presence of AOA, only 20% inhibition of glucose oxidation
occurred. This poses the question as to whether other shuttles, not involving transaminase
reactions, can contribute to glucose oxidation in the heart as such as/OAA and L-LAC/PYR
shuttles (see below).
4. Phosphoenolpyruvate (PEP) Transport in Mitochondria
In the last century, PEP transport in and from mitochondria has been poorly investigated.
The first evidence of PEP capability to cross the mitochondrial membrane was provided in
1967 by Gamble and Mazur who showed that citrate addition to rabbit liver mitochondria
(RbLM) resulted in the PEP appearance outside mitochondria [53]. The authors concluded
that “This could be due to a specific positioning or compartmentation of the reaction or,
alternatively, to a specific permeability of the membrane”. As reported in [9], Drahota et al.
[54] and Wiese et al. [55] showed that PEP can enter mitochondria. A revolution in PEP
mitochondrial transport and metabolism occurred in the first decade of the third millennium,
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when PEP transport in mitochondria was investigated, with evidence of the existence of the
mitochondrial PK both in plant [56] and mammalian mitochondria [57,58].
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In the first case, coupled mitochondria isolated from Jerusalem artichoke tubers (JAM) searchmenu
were used and an investigation was made utilizing mainly the dynamic approach.
Interestingly, as opposed to the expected increase in in NAD(P)H fluorescence, (an increase
due to the NAD(P)+ reduction arising from the via intramitochondrial metabolism of the newly
synthesized PYR via a mitochondrial by PK), upon PEP addition to JAM, intramitochondrial
NAD(P)H oxidation was found due to the activation of the alternative oxidase (AOX)
determined by the capability of the AOX inhibitors, both propyl-Gallate and salicyl-hydroxamic
acid (SHAM), AOX inhibitors, to inhibit this process. Nonetheless, in spite of the four steps
involved
 in this process, i.e., PEP uptake, the PK reaction, AOX activation by PEP and 
pyruvate with the consequent intramitochondrial NAD(P)H oxidation, the decrease in
fluorescence proved to mirror the PEP uptake as shown by applying control strength criterion.
It was found that:
i. PEP can be metabolized by JAM by virtue of the presence of the mitochondrial PK, shown
both immunologically and functionally, located in the inner mitochondrial compartments,
and distinct from the cytosolic PK (shown by the different pH and inhibition profiles).
ii. PEP uptake into JAM occurs in a proton compensated manner, in a carrier-mediated
process.
ii. The PEP addition resulted in PYR and ATP production, as monitored via HPLC, with their
efflux into the extramitochondrial phase as detected fluorimetrically (see above). Such an
efflux occurs via the putative PEP/PYR and PEP/ATP antiporters that differ from each other
and from the PYR and the adenine nucleotide carriers, based on the different sensitivities
to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of
both and ATP. The appearance of citrate and OAA outside mitochondria was also found
because of PEP addition.
The PK existence in mitochondria was confirmed in pig liver mitochondria [57,58]. The
researchers took advantage of a bioinformatic suggestion: as a result of a search for
‘‘mitochondrial pyruvate kinase” in the NCBI genome database, a gene (LOC100154270)
coding for a protein similar to PK which that is predicted to have a mitochondrial localization
by Target P 1.1 analysis (reliability class = 3) was found in the Sus scrofa genome database.
However, the PK occurrence was shown by monitoring photometrically the PK reaction in
solubilized mitochondria with either PEP or ADP used as a substrate. In distinction with the
cytosolic isoenzyme, the mitochondrial PK showed a sigmoidal dependence on either PEP or
ADP concentrations. The occurrence of the mitochondrial PK was confirmed by
immunological analysis. Titration with digitonin showed that mPK is restricted to the matrix.
PEP addition to mitochondria that resulted in a reduction in the intramitochondrial NAD(P)+
was inhibited by either the non-penetrant thiol reagent mersalyl or by arsenite, an inhibitor of
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the pyruvate dehydrogenase complex.
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Citrate/OAA appearance outside mitochondria also occurred as a result of PEP addition
to PLM. Taken together, these findings support a role for PEP itself in triggering fatty acid searchmenu
synthesis via its mitochondrial metabolism. This process is described in the paper by Di
Ciaula et al. [59].
These findings shone new light to previous results previously published in the Pasqualina
Pierro Ph.D. thesis work (1991–1995) and only as reported in a meeting [60] and in [12]. We
offer these experiments as a matter of discussion for further investigation.
Fluorimetric investigation of the change in the redox state of the intramitochondrial
pyridine nucleotides brought about by the PEP addition was made (Figure 7). Mitochondria
were
 isolated from rabbit kidney mitochondria (RbKM) and RKM that differ from one another

with respect to the localization of the PEP carboxykinase, which is present in both cytosol and
mitochondria of RbKM but is absent in RKM. In both cases, PEP proves to enter mitochondria
as shown by the increase in the intramitochondrial NAD(P)H fluorescence. The addition of
arsenite (1 mM), an inhibitor of the pyruvate dehydrogenase complex (PDH), resulted in
complete inhibition of the NAD(P)H formation. This shows that the fluorescence increase is
due to the oxidation via PDH of the PYR newly synthesized from imported PEP via the
mitochondrial PK.
Figure (/)7. Phosphoenolpyruvate (PEP) can enter both rabbit and rat kidney
mitochondria. From [12]. From Aracne Editrice with permission 2021. Fluorimetric
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investigation was made of the change in the redox state of the intramitochondrial
pyridine nucleotides caused brought about by the addition of PEP. Rabbit and rat kidney
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mitochondria (RbKM and RKM, respectively) (2 mg protein) were suspended at 25 °C in
2 mL of standard medium consisting of 15 mM KCl, 1 mM MgCl2, 100 mM TRIS-HCl,
pH 7.4. and the redox state of intramitochondrial pyridine nucleotides was followed
fluorimetrically (λex = 334 nm/λem = 456 nm) as a function of time. First, either RbKM or
RKM were incubated for 3–5 min with FCCP (1.25 mM) and then rotenone was added
(2 mg) (not shown). At the arrow, PEP (2.5 mM) was added either in the absence or
presence of α-cyano-4-hydroxy-cynnamate (CCN−) or arsenite (0.01 and 1 mM,
 respectively). The rate of fluorescence increase, measured as the tangent to the initial 
part of the progress curve, is expressed as nmol of intramitochondrial NAD(P)+
reduced/min × mg protein. PEP uptake by mitochondria was prevented in the presence
of either α-cyano-4-hydroxycynnamate (CCN− 10 μM), or benzylmalonate (5 mM) (not
shown), which can inhibit a variety of carriers. These simple experiments have
produced the first evidence that mammalian mitochondria contain their own PK and
showed that mitochondrial PEP transport occurs in a carrier-mediated manner.
At that time PK was considered only a cytosolic enzyme and no further investigation on
this issue was pursued. However, in light of the metabolite transport paradigm [9], postulating
that net carbon uptake by mitochondria is accompanied by the efflux of certain newly
synthesized compounds, an investigation was made to ascertain whether or not the addition
of PEP to RbKM and RKM could result in the efflux of pyruvate and ATP, the products of the
mitochondrial PK reaction (Figure 8A,B). Either RbKM or RKM were suspended at 25 °C in 2
mL of standard medium consisting of 15 mM KCl, 1 mM MgCl2, 100 mM TRIS-HCl, pH 7.4 in
the presence of either NADH (0.2 mM) (A and A1, C and C1, D and D1) or NAD(P)+ (0.2 mM)
(B and B1, E and E1) and absorbance at 334 nm (A334) was continuously monitored.
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 
Figure 8. Appearance of pyruvate (PYR) (A), ATP (B), oxaloacetate (OAA) (C), citrate
(CITR) (D), and malate (MAL) (E) in the extramitochondrial phase space induced by
PEP addition to rabbit kidney mitochondria (RbKM) and appearance of PYR (A1), ATP
(B1), OAA (C1), CITR (D1), and MAL (E1) in the extramitochondrial phase space
induced by the addition of PEP, ADP, MAL, and fumarate (FUM) to rat kidney
mitochondria (RKM). From [12]. With permission from Aracne Editrice, 2021.
At the times indicated by the arrows the pyruvate detecting system (PYR D.S.) (A, A1),
the ATP detecting system (ATP D.S.) (B, B1), the OAA detecting system (OAA D.S.) (C, C1),
the citrate detecting system (CITR D.S.) (D, D1), and the malate detecting system (MAL D.S.)
(E, E1) were added followed by PEP (2.5 mM), (A–E, A1–E1) ADP (0.05 mM) plus P1,P5-
di(adenosine-5′) pentaphosphate (Ap5A) (0.01 mM), added to inhibit the adenylate kinase (B,
B1), MAL (1 mM) (C, C1) or FUM (1 mM) (E, E1).
In all cases, use was made of the metabolite/compound detecting systems described in
Table 2. The rate of NADH oxidation/NADP+ reduction was measured as a tangent to the
initial part of the progress curves and expressed as nmol NADH oxidized/NADP+ reduced/min
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× mg mitochondrial protein.
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As a result of PEP (2.5 mM) addition to RKM both PYR and citrate were detected in the
extramitochondrial space. This contrasted with what was found with RbKM, where no efflux of searchmenu
ATP, OAA, or malate was found, as shown by the constancy of the unchanged absorbance at
334 nm in the presence of their detecting systems. As a control, RKM was tested for their
ability to export ATP, OAA, and malate upon the addition of ADP (0.05 mM), malate (1 mM),
and fumarate (1 mM), most likely via the ADP/ATP, the MAL/OAA and the FUM/MAL
antiporters, respectively.
Both, to gain further insight into the permeability of RKM to PEP and to investigate
whether or not PEP can be synthesized in the mitochondrial matrix via PK and exported
outside
 RKM, PYR (2.5 mM) was added to mitochondria. Since the appearance of PEP 
outside the mitochondria cannot be continuously monitored, neither via PK and L-LDH, due to
the presence of PYR, nor as ATP formation via the ATP D.S., due to the presence of ADP,
which can drive both ATP synthesis and its export, an enzymatic assay was made to
ascertain the PEP presence. PEP was found in the supernatants (about 6 nmol/mg
mitochondrial protein) with its amount increasing up to 16 nmol/mg mitochondrial protein due
to the presence in the incubation medium of arsenite (1mM), which can prevent PYR
oxidative decarboxylation via PDH, making more PYR available for PEP synthesis.
To confirm the capability of externally added PYR to cause PEP efflux from mitochondria
a different isotopic approach was required. Thus [14C]-PEP-loaded mitochondria were used.
PYR was added at increasing concentration and after 1 min incubation, the mitochondrial
suspension was centrifuged and the supernatants were assayed for [14C]-PEP presence. In
both cases, PYR/14C-PEP exchange was found to occur with saturation kinetics as shown by
studying the dependence of the rate of PEP efflux as a function of increasing PYR
concentrations (Figure 9). Vmax values were about 9.5 and 7.3 nmol/min × mg protein,
respectively and Km values were 0.85 and 1.4 mM for RKM and RbKM, respectively. Figure
10 describes the possible scenario derived from the above experiments.
9. The dependence of the rate of the pyruvate/[14C]-phosphoenolpyruvate (PEP)

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exchanges in either rat (A) or rabbit (B) kidney mitochondria on the external pyruvate
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concentration. From [12]. With permission from Aracne Editrice, 2021. [14C]-PEP-
loaded mitochondria (1 mg protein) were incubated at 4 °C in 1 mL of a standard
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medium consisting of 15 mM KCl, 1 mM MgCl2, 100 mM TRIS-HCl, pH 7.4 plus 1 µg of
µg rotenone, 3 µg oligomycin in the presence of 5 mM arsenite and 10 mM oxalate.
After 1 min incubation, the assay was started with pyruvate, at the concentrations
indicated, and was stopped after 6 seconds by the addition of 10 mM phenylsuccinate.
The exported 14C-PEP was assayed as reported above. Both RKM and RBKM were
loaded with 14C-PEP and its efflux measured according to the stop inhibitor method.
 
Figure 10. The role of the mitochondrial pyruvate kinase in gluconeogenesis in rat
kidney. The following scenario is proposed: pyruvate (PYR) derived from L-lactate and
amino-acids enters mitochondria via its own carrier; once inside the matrix via the
mitochondrial pyruvate kinase (PK) phosphoenolpyruvate (PEP) is synthesized, the
reaction being made possible by the removal of the newly synthesized PEP which is
exported in exchange with PYR via the putative PYR/PEP antiporter.
The PEP-mitochondria affair remains open. However, it must be stressed that PEP
transport and metabolism in mitochondria—as well as the mitochondrial PK—have not been
given any consideration in several papers published in the second decade of this century.
These include a paper by McCommis and Finck [61] paper dealing with mitochondrial PYR
transport, one by Cerdan (2017) [62] in which twenty-seven years of cerebral PYR recycling
is reported, and another by Chinopoulos [63], in which biochemical pathways in which twenty-
seven of cerebral years biochemical pathways connecting glucose and other metabolites to
PYR and L- or D-LAC are described. A revision of the PEP metabolism in the light of old and
new achievements findings appears to be necessary.
Interestingly, in skeletal muscle, trioses are generated via reversal of PK and L-LAC
contributes to glycerol and glycogen via reversal of PK [64].
5. L-Lactate Transport in Mitochondria

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5.1. The L-Lactate History
Although the L-LAC history begins more than a century zoom_out_map
ago, the role of mitochondria in
L-LAC metabolism has received recognition only at the outset of the third millennium. Notably,
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in the past two decades, authors writing the L-LAC history had different scientific
backgrounds. Physiologists consider L-LAC as a major product of exercise, neuroscientists
approach L-LAC as an important oxidative substrate for brain energy metabolism, while
others are experts in mitochondrial research involved in the role of mitochondria in the L-LAC
metabolism. The consideration of L-lactate biochemistry, when approached from different
perspectives, explains why limited knowledge of mitochondrial bioenergetics would lead to
potential misunderstandings (see ref. [7]).
 
Until the mid-1980s, L-LAC was considered to be a waste product rather than an energy
source for a variety of cells, and L-LAC was postulated to be taken up and oxidized (see [10]).
Thereafter, the role of mitochondria in L-LAC metabolism has been demonstrated in
spermatozoa, liver, heart, skeletal muscle, brain, plant, and yeast mitochondria. As for the
latter, the history of the L-lactate–mitochondrial affair up to 2008 has been outlined in a mini-
review by Passarella et al. [10]. Figure 11 describes the status of the L-lactate–mitochondria
affair in 2008.
Figure 11. The mitochondrial metabolism of L-lactate. From Passarella et al. From [10].
With permission from John Wiley and Sons, 2021.
(A) The mitochondrial metabolism of L-lactate in potato tuber. The sequence of events
involved in mitochondrial metabolism of L-lactate (L-LAC) is envisaged as: uptake into
mitochondria of L-LAC, synthesized in the cytosol by anaerobic glycolysis, perhaps via the L-
LAC/H+ symporter; oxidation of the L-LAC to PYR by the mL-LDH located in the inner
mitochondrial compartment; activation of alternative oxidase (AOX) by the newly synthesized
PYR, oxidation of the intramitochondrial NAD(P)H via AOX with efflux of PYR via a putative L-
LAC/PYR antiporter and the oxidation of cytosolic NADH in a non-energy-competent L-

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LAC/PYR shuttle. PYR conversion could also occur to AcetylCoA and malate via pyruvate
dehydrogenase and malic enzyme, respectively.
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(B) The mitochondrial metabolism of L-lactate in the liver. Externally added L-LAC can searchmenu
enter RLM where it is oxidized by the mitochondrial L-LDH. L-lactate can cause efflux in the
extramitochondrial phase of PYR and OAA newly synthesized in the mitochondrial matrix via
mL-LDH and pyruvate carboxylase. The metabolite efflux occurs by virtue of the occurrence
of three carriers for L-LAC transport in mitochondria: the L-LAC/H+ symporter and the L-
LAC/PYR and L-LAC/OAA antiporters. The LAC/PYR antiporter accounts for the LAC/PYR
shuttle which transfers reducing equivalents from the cytoplasm to the mitochondrial
respiratory chain. The L-LAC/OAA antiporter accounts for novel gluconeogenesis. OAA and
PYR
 (via the pyruvate dehydrogenase) could also fill up the Krebs cycle intermediate pool. 
(C) The mitochondrial metabolism of L-lactate in Saccharomyces cerevisiae. Externally
added L-LAC can enter mitochondria via a putative L-LAC/H+ symporter. In mitochondria, an
NAD-dependent mL-LDH exists. Moreover, in the intermembrane space L-LAC is oxidized to
PYR with reduction in cytochrome c by a flavin mitochondrial L-lactate:cytochrome c
oxidoreductase in an energy competent manner. Abbreviations: AcCoA, acetyl-CoA; AOX,
alternative oxidase; GNG, gluconeogenesis; L-LAC, L-lactate; MAL, malate; OAA,
oxaloacetate; PYR, pyruvate; R.C., respiratory chain; transport and oxidation processes the
existence of which has not yet been confirmed. Enzymes: a, cytosolic L-LDH; b, mitochondrial
L-LDH; c, malic enzyme; d, pyruvate dehydrogenase; e, pyruvate carboxylase; f, L-
lactate:cytochrome c oxidoreductase (Cyb2p). Mitochondrial carriers: 1, L-LAC/H+ symporter;
2, L-LAC/PYR antiporter; 3, L-LAC/OAA antiporter.
Interestingly, de Bari et al. [65] provided evidence for the presence of an intermembrane
L-lactate oxidase that generates H2O2 sufficient to activate the known ROS response
elements, signaling mitochondrial and other adaptations to exercise. That work provides a
possible mechanism by which L-LAC generation in muscle exercise participates in the
feedback loop. L-LAC generation in exercise leads to adaptations facilitating high rates of
lactate disposal in exercise. It is worth noticing that the putative L-LAC oxidase could be a
candidate to the new role proposed for L-LAC as “lactormone”, i.e., in Brooks’ term [66] as a
cell-signaling molecule that is involved in the adaptive response to exercise. In 2015, Paventi,
Lessard, Bailey, and Passarella [67] found that in boar sperm capacitation L-LAC, but not
PYR can contribute to mitochondrial membrane potential increase as monitored via safranine
fluorescence, this both strongly suggesting that L-LAC is the ultimate product of glycolysis
and confirming that L-LAC must enter mitochondria to be oxidized by the mL-LDH (see also
[10]).
We will consider some aspects of the mitochondrial L-LAC transport and metabolism in
neuronal and cancer cells not already considered in [10].
5.2. The L-Lactate Mitochondrial Metabolism Plays a Major Role in Neuronal Cells
A better understanding of the role of L-LAC in neuronal energy metabolism and in

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particular in mitochondrial energy metabolism (see [10]) requires some attention. In fact, for
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almost a century this molecule was considered just a waste product of the brain during
hypoxia. The lactic acidosis hypothesis of delayed neuronal damage post cerebral ischemia searchmenu
was proposed in 1981 [68], and the researchers quickly explained the cellular mechanism
that leads to this phenomenon, i.e., the fall in pH post-ischemia leads to delayed neuronal
damage and this drop is due to the ischemic production of L-LAC. Upon attempting to
establish an in vitro model of cerebral ischemia by showing that lactic acidosis would worsen
ischemic neuronal damage, the investigators were surprised to find that acidic pH provided
slight protection, while L-LAC provided dramatically better protection [69].
Consequently, a seminal paper demonstrated the ability of neuronal tissue to utilize L-
LAC
 as its sole oxidative energy substrate to maintain neuronal function [70]. This finding did

not sit well with the prevailing dogma of the time regarding glycolysis, L-LAC, and oxidative
energy metabolism leading to a long-lasting debate that has not subsided to this day.
Nevertheless, the above-mentioned in vitro model system allowed the further exploration of
the role of L-LAC as an oxidative substrate of neuronal tissue main substrate for oxidative
energy metabolism. By 1994, Izumi et al. [71] confirmed this finding. Similarly, Larrabee
[72,73] provided further support for neuronal oxidative utilization of L-LAC. L-LAC was shown
to be the obligatory energy substrate for the recovery of neuronal function from
hypoxic/ischemic insult [74,75]. Using in vivo recording in the rat brain, Hu and Wilson [76]
demonstrated that fluctuations in the levels of extracellular L-LAC and oxygen levels are
coupled to neuronal activity. An increase in L-LAC output upon neuronal excitation in vitro
was shown to serve the need for increased energy demands of excited neurons [77]. In
contrast, the blockade of L-LAC transport via the monocarboxylate transporter 1 (MCT1)
exacerbates delayed neuronal damage in a rat model of cerebral ischemia [78]. This finding
established L-LAC as the preferential oxidative energy substrate when ATP stores dwindle to
the point that glycolytic glucose phosphorylation is unattainable and possibly even under
normal physiological conditions. In 2003, Smith et al. [79] showed that L-LAC is the preferred
fuel for human brain metabolism in vivo and Dalsgaard et al. [80] demonstrated that reduced
cerebral metabolic ratio in exercise reflects L-LAC metabolism rather than accumulation in the
human brain. Thus, in 2006 L-LAC was suggested to be the ultimate cerebral oxidative
energy substrate [81]. By 2007, it was demonstrated that L-LAC, not PYR, is the neuronal
aerobic glycolysis end product in vitro [81,82]. In that study relatively specific LDH inhibitors
were used to inhibit either the L-LAC-to-PYR reaction or the PYR-to-L-LAC one, enabling the
investigators to determine that L-LAC, rather than PYR, is the substrate that is being oxidized
intra-mitochondrially by LDH.
During the preparation of the 2006 review [81], a thorough search of older literature from
the 1920s and 1930s, rediscovered a throve of studies, most of which were performed by one
group of biochemists. Those investigators demonstrated and were aware of the ability of brain
tissue preparation to make lactic acid disappear in the presence of oxygen, a process the
investigators interpreted as a mechanism of lactic acid clearance

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[83,84,85,86,87,88,89,90,91,92].
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The prevailing dogma at the time had been that lactic acid is a waste product to be
cleared. It is important to clarify that Krebs and Johnson were not sure about their suggestion searchmenu
that PYR is the mitochondrial substrate of the TCA cycle [93,94,95], but that suggestion was
the one responsible for the decision by the elucidators of the glycolytic pathway to place PYR
as its final aerobic product. This digging might suggest that biochemical archeology could still
be a source of inspiration.
That L-LAC can be a neuronal energy source was also shown by O’Brien et al. [95] and
Wyss et al. [96].
A short commentary [97] weighed in on the results published by Larsen et al. [98]. These
investigators
 described their attempts to elucidate the mechanism behind the observed 
exercise-induced reduction in the cerebral metabolic ratio (CMR) as measured in healthy
human subjects. They no longer used the accepted definition of CMR as O2/glucose, but
rather focus instead on the more accurate definition of CMR, i.e., O2/(glucose + ½ L-LAC), to
make their estimates. They clearly show that as their subjects approached the maximal work
load and exhaustion under control conditions, the CMR [O2/(glucose + ½ L-LAC)] fell from the
expected value of approximately 6 to about 3. When the same measurement was done for
CMR (O2/glucose), its value did not change significantly throughout the experimental period.
Concomitantly, the a–v differences for glucose and oxygen, even at the maximum workload,
increased only slightly in comparison to the values at rest. In contrast, the a–v differences for
total carbohydrates (CHO) at maximal workload were significantly higher than the values at
rest, an increase that could be attributed almost entirely to the significantly higher a–v
difference for L-LAC. Based on the results of Larsen and colleagues, Schurr concluded that
L-LAC is a major and crucial player in the normal function of both brain and muscle [97]. In
2011, it was shown that aerobic production and utilization of L-LAC satisfy increased energy
demands upon neuronal activation in hippocampal slices and provide neuroprotection against
oxidative stress [99]. The authors analyzed, among others, the results of the study by Hu and
Wilson [76] who measured glucose, L-LAC, and oxygen level in brain tissue in vivo during
rest and stimulation. The analysis demonstrated that, during continuous stimulation, brain
tissue consumes, oxidatively, gradually more L-LAC and less glucose, a process that allows
for a more efficient ATP production to support neuronal activation induced by such
stimulation. Moreover, in vitro experiments carried out by Schurr and Gozal [99] indicated that
the production of reactive oxygen species (ROS) in response to the presence of the
excitotoxic neurotransmitter, glutamate was reduced significantly when L-LAC was the sole
energy substrate. PYR—as the sole energy substrate—could not reduce ROS production.
Despite the continuous accumulation of studies supporting L-LAC’s major role in brain energy
metabolism, doubts about their validity are persisting, even today. In a review [100] the
authors opined on the persistence of doubt among scientists regarding L-LAC’s role as a
major oxidative substrate for energy metabolism in the brain and elsewhere and offered that a
“habit of mind” may explain the persistence of that doubt. Considering the proposed function
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of L-LAC in energy metabolism, the accuracy of current methods and techniques used to
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measure brain tissue metabolic rates of glucose only, not taking into account the contribution
of L-LAC to these measurements was questioned.
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Clearly, there is ample evidence to support the concept that brain L-LAC is the glycolytic
end product, both aerobically and anaerobically, and hence the main oxidative substrate for
mitochondrial TCA.
Evidence that L-LAC transport and metabolism can occur in brain mitochondria was
demonstrated for the first time in 2007 when mitochondria from cerebellar granule cells were
shown to metabolize externally added L-LAC [14]. This has been confirmed [95] and where
the occurrence of LDH in the mitochondria of an astrocytic cell line was shown. In 2008
Hashimoto
 et al. [66] proposed that neurons contain a mitochondrial lactate oxidation
complex that has the potential to facilitate both intracellular and cell–cell lactate shuttles in the
brain (see below).
5.3. The L-Lactate Mitochondrial Metabolism Plays a Major Role in Cancer Cells
In cancer, L-LAC is the ultimate product of glycolysis (Warburg effect), however, until
2010 its metabolism has not been investigated in detail: in the words of Kennedy and
Dewhirst [101] “The implications and consequences of L-LAC utilization by tumors are
currently unknown; therefore, future research is needed on the intricacies of tumor
metabolism”. No mitochondrial metabolism was proposed until 2010 when de Bari et al. [20]
published a paper in which showed that “L-LAC metabolism can occur in normal and cancer
prostate cells via the novel mitochondrial L-LAC dehydrogenase”. By using, mainly, the
dynamic approach described above, they showed that L-LAC can be transported into
mitochondria isolated from both normal (PTNA) and cancer cells (PC3 cells) in a carrier-
mediated manner via the putative L-LAC/H+ symporter, inhibited by the thiol reagent mersalyl.
Inside mitochondria L-LAC is oxidized by the mL-LDH, producing PYR. Normal and cancer
cells were found to differ from one another with respect to mL-LDH protein level and activity,
being the enzyme more highly expressed and of higher activity in cancer cells. Such a
conclusion was confirmed by Hussien and Brooks who found differences in mitochondrial
LDH and MCT isoform expression in normal breast cancer and breast cancer cells [102].
Moreover, the kinetic features and pH profiles of the PC3 mL-LDH also differ from those of
the PNT1A enzyme, this suggesting the occurrence of separate isoenzymes. Considering the
poor oxygen consumption and since fatty acid oxidation is the bioenergetic dominant pathway
in the prostate, L-LAC metabolism was suggested to lead to citric cycle anaplerosis to give
OAA via pyruvate carboxylase, activated by acetyl-CoA. Citrate could be then formed to be
used essentially in fatty acid synthesis in PC3 cells and exported in the extracellular fluid in
the PNT1 cells. Two years later, confirmation that L-LAC uptake can trigger metabolic traffic
from cytosol to mitochondria and vice versa was found in Hep G2 cells: occurrence of the L-
LAC/PYR shuttle (see [9,10]) and the appearance outside mitochondria of OAA, malate and
citrate arising from L-LAC uptake and metabolism together with the low oxygen consumption
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and membrane potential generation were found thus establishing an anaplerotic role for L-
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Lactate in Hep G2-M for instance in fatty acid synthesis [21]. As emphasized above, the same
conclusion was proposed 4 years later for other cancer cells. It was found that Hep G2 cell searchmenu
mitochondria (Hep G2-M) possess an mL-LDH restricted to the inner mitochondrial
compartments as shown by immunological analysis, confocal microscopy and by assaying
mL-LDH activity in solubilized mitochondria [21]. Cytosolic and mitochondrial L-LDHs were
found to differ from one another in their saturation kinetics. The capability of L-LAC to enter
mitochondria was shown by measuring the increase in NAD(P)H fluorescence which takes
place as a result of L-LAC addition and by monitoring the mitochondrial swelling in
ammonium L-LAC solution. Interestingly, in the same experiment, PYR proved to be a non-
penetrant
 metabolite, this suggesting the impossibility that the Cori cycle could occur in these

cells. Accordingly, Passarella and Schurr [103] published “L-lactate transport and metabolism
in mitochondria of Hep G2 cells-the Cori Cycle revisited”, in which, due to the lack of the PYR
carrier activity in cancer mitochondria, it is proposed that gluconeogenesis in Hep G2 cells
depends on L-LAC mitochondrial transport, where OAA is formed and exported for
gluconeogenesis likely via the L-LAC/OAA antiporter. Recently, it was proposed to include the
mitochondrial metabolism of L-LAC in cancer metabolic reprogramming [104].
5.4. A Quick, Easy Protocol to Investigate the L-Lactate Transport and Metabolism in Muscle
Mitochondria
To ascertain the occurrence of L-LAC transport and metabolism in coupled muscle
mitochondria a simple protocol to be used was proposed by Passarella and colleagues who
published “The mitochondrial L-lactate dehydrogenase affair” [22] in which the occurrence of
an L-LDH and L-LAC metabolism in isolated rabbit gastrocnemius mitochondria was shown.
Use was made of the experimental strategy required to show if and how L-LAC can enter
mitochondria for subsequent metabolism. Rabbit gastrocnemius muscle was rapidly isolated
(5–10 min) following killing the animal and immediately placing it in ice-cold KCl medium (0.1
KCl, 50 mM Tris-HCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, pH 7.5). Mitochondria (RGM)
were isolated with the exclusion of protease treatment (which proved to result in mitochondrial
uncoupling and damage of the mitochondrial carriers, thus making any study of mitochondrial
transport impossible) and immediately checked for their intactness by assuring that no
reduction in absorbance at 334 nm occurred upon NADH addition. m-L-LDH activity was
found in RGM solubilized with 0.1% Triton X-100 (TX-100) as the decrease in absorbance of
NADH after PYR addition. No absorbance change should occur when PYR is added to intact
mitochondria; clearly indicating that m-L-LDH is in the inner mitochondrial compartment.
Surprisingly, such a simple assay was not reported in a paper in which the existence of m-L-
LDH was argued against because “the distribution of L-LDH activity, among the fractions
paralleled that of PK” [105]. Unfortunately, the occurrence of a mitochondrial PK was later
shown by Pizzuto et al. [57,58]. In another study, L-LDH activity was considered to be
negligible [106]. Thus, we opine that it is easy to dismiss that m-L-LDH is in the outer
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mitochondrial membrane/intermembrane space: no NADH oxidation occurs when PYR is
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added to purified mitochondria, whereas treatment of mitochondria with TX-100 results in
NADH oxidation via Complex I with rate increased by the addition of PYR, which reacts with searchmenu
NADH via m-L-LDH.
The existence of an m-L-LDH localized in the inner mitochondrial compartment was
simply established by showing the ability of externally added L-LAC to reduce the
intramitochondrial NAD+. The involvement of L-LDH in this process was confirmed by its
inhibition with oxamate, an inhibitor of LDH.
Consistently with the spectroscopic outcome, a static immunological analysis has shown
that mitochondria free of cytosolic contamination (no tubulin, a marker of the cytosolic
fraction)
 contain a protein recognized by the L-LDH antibody. As noted above, such a static

measurement by itself cannot afford one to determine in which mitochondrial compartment
mL-LDH is located.
One wonders why the investigators who have firmly denied the existence of
mitochondrial L-LDH did not carry out these simple experiments. Perhaps their view was
colored by the mistaken belief, based on incorrect thermodynamic arguments, that
mitochondria cannot import L-LAC. Indeed, both Rasmussen et al. [105] and Sahlin et al.
[107] argue that the idea of L-LAC conversion to PYR inside mitochondria is not feasible
based on thermodynamic principles. They point to a much higher reduction in the
NAD+/NADH redox couple inside mitochondria; so much higher in fact that it would
theoretically eliminate the possibility of L-LAC to PYR conversion. Sahlin et al. [107] went on
to suggest that if m-L-LDH was present in the mitochondrial matrix, it would lead to a futile
cycle in which PYR would be reduced to L-LAC in mitochondria and vice versa in the cytosol,
oxidizing mitochondrial NADH and finally removing the driving force for the electron transport
chain. However, given that in brain mitochondria the NAD+ concentration is 8–20-fold higher
than that of NADH and that PYR is actively oxidized via pyruvate dehydrogenase, it was
suggested [14] that m-L-LDH in vivo essentially catalyzes L-LAC oxidation. Ultimately, the
removal of the oxidation product by carrier-mediated transport and mitochondrial metabolism
overcomes any thermodynamic difficulty. Hence, those results are consistent with the
postulate/proposal of Schurr et al. [81,82] that L-LAC is the only major product of cerebral
glycolysis and that it can be metabolized inside mitochondria. On the other hand, glucose
oxidation to L-LAC is expected to occur when oxidative phosphorylation is reduced since
citrate and/or other citric acid cycle intermediates are required outside mitochondria for
anabolism to occur (e.g., see [21]). Under such conditioned anaerobic glycolysis is expected
to provide ATP and L-LAC in mitochondria to play an anaplerotic role and/or to be transferred
to other cells in the intercellular shuttle.
The results of these studies clearly require us to revise the standard, dogmatic view of
glucose metabolism via glycolysis and oxidative phosphorylation. Surprisingly, the
overwhelming evidence for the location of mL-LDH inside mitochondria has not yet been
accepted by the “mL-LDH non-believers”. Additional possible explanations for the failure of
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the “m-L-LDH non-believers” to monitor L-LAC mitochondrial metabolism in muscle can be
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proposed. In those studies, skeletal muscle mitochondria were investigated. It is our opinion
that the investigators involved were unable to isolate coupled mitochondria, a task, which is searchmenu
not easy, where skeletal muscle samples are concerned, and that they may not be careful
enough in selecting reaction media and the use of inhibitors at the right concentrations. For
instance, the failure to measure oxygen consumption as a result of L-LAC addition to skeletal
muscle mitochondria [108] could be due to the presence of 5 mM MgSO4 in the medium used
to prepare isolated skeletal muscle mitochondria, where sulfate is known to enter
mitochondria and to cause efflux of intramitochondrial phosphate, malate, and succinate
[109].
 Moreover, 60 mM lactobionate included in the medium used to measure oxygen uptake 
by mitochondria is expected to prevent L-LAC uptake due to its chemical structure and high
concentration. Finally, no control samples were reported of c-L-LDH and m-L-LDH sensitivity
or insensitivity to 60 mM lactobionate. Notice that 60 mM lactobionate was used in a paper in
which lactate oxidation was proposed to occur outside mitochondria [110]. Of course, it is
impossible to mimic cytosol totally with the medium used for in vitro experiments, however,
control must be made that the absence of L-LAC metabolism is not dependent on the
composition of the media used in the experiments as well as on the mitochondria isolation.
5.5. Achievements in the L-Lactate-Mitochondria Affair
Although the role of L-LAC in energy metabolism has been revisited at the beginning of
this century, whether and how mitochondria can take up and metabolize L-LAC is still ignored
in the literature concerning both normal and cancer cells. Surprisingly enough, in many
papers dealing with mitochondria and cancer published in the last decade
[4,8,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,13
1,132] no mention of the L-LAC metabolism in mitochondria was made this confirming that
incorrect use of the Web database can result in incorrect information for the readers [133] and
raising serious doubts about the reliability of incomplete information.
On the other hand, recently, Fulghum et al. [134] concluded that mLDH does not
“contribute to cardiac bioenergetics in mice”. No mention of the papers of the authors of this
review was made. Contrarily, in 2020 Young et al. report [135] “Using the guide supplied by
Passarella et al., [22] we counter the conclusions drawn by Fulghum et al. and demonstrate
that mitochondria oxidize lactate”. It was a great surprise that in 2020 Borst reported “I concur
with the sceptics and disregard matrix mitoLDH until proven wrong by rigorous experiments”
[47]. Indeed “Quandoque bonus dormitat Homerus”! (even the great Homer sometimes
dozes). An even greater surprise was that the paper containing this sentence has been
published without any citation of the work of the authors of this review.
Therefore, in this review we will try to provide definitive answers to several specific
questions, to offer closure to the L-LAC-mitochondria affair.
- Question 1: Do L-LAC transport and metabolism occur in mitochondria? The answer

(/)
here is yes, they do. The existence of an mL-LDH was definitively confirmed in mammalian,
of mL-LDH in the zoom_out_map

plant, and yeast mitochondria (see [103]) with its existence being finally recognized by the
inclusion
MitoCarta: searchmenu
(http://www.broadinstitute.org/pubs/MitoCarta/index.htrnl
(https://www.broadinstitute.org/pubs/MitoCarta/index.htrnl)) (accessed 20 November
2021).
- Question 2: Do L-LAC and PYR enter mitochondria via the same carrier? The answer
here is no since there are separate carriers!
The mechanism by which L-LAC enters mitochondria has often not been considered or it
is assumed that L-LAC enters mitochondria via the PYR transporter. However, in RLM it was
shown
 that two separate carriers transport PYR and L-LAC into mitochondria. Although α-
cyano-hydroxycinnamate can inhibit the uptake of both PYR and L-LAC, the PYR carrier is
inhibited at a concentration (25 µM) at which no inhibition of L-LAC transport occurs.
Moreover, other antiports for L-LAC have been shown to occur: comparison made among the
inhibition profiles of L-LAC transport/transporters due to non-penetrant compounds shows
that the L-LAC carriers, (the L-LAC/H+ symporter, the L-LAC/PYR, and L-LAC/OAA
antiporters), the PYR carrier and other carriers, including those that transport D-LAC, all differ
from one another [19].
- Question 3: In which of the mitochondrial compartments is L-LDH located?
The mitochondrial L-lactate is in the mitochondrial matrix. That L-LAC enters
mitochondrial matrix where is oxidized by the mitochondrial L-LDH has been shown using
many experimental approaches:
(1) Swelling measurements exhibiting the stereospecificity of the process and the
inhibition of swelling found due to non-penetrant. Obviously, a carrier-mediated transport itself
postulates that L-LAC is metabolized inside mitochondria.
(2) Measurements of an increase in the redox state of the intramitochondrial pyridine
nucleotides found because of the addition of L-LAC to mitochondrial samples in the absence
of external NAD+ indicate that mitochondrial metabolism occurs inside the organelles via the
NAD+-dependent mL-LDH.
(3) Measurements of oxygen consumption by coupled purified mitochondria upon L-LAC
addition inhibited by both rotenone, inhibitor of complex I, and oxalate/oxamate, inhibitors of
L-LDH.
(4) Proton efflux and the increase in membrane potential were found because of L-LAC
uptake and metabolism. Conversely, proton uptake occurs because of L-LAC addition to
mitochondria previously treated with an inhibitor cocktail used to prevent any energy
metabolism, due to the L-LAC/H+ symport.
(5) Measurements show the efflux of a variety of metabolites newly synthesized inside
mitochondria due to externally added L-LAC.
Last, but not least, (a) enzymatic assays (b) immunological analysis, and (c) confocal
(/)
fluorescence microscopy can be used: (a) allows for initial information about enzyme kinetics
zoom_out_map
features; (b) and (c) can reveal the existence of m-L-LDH even if inactive but have the
limitation that they are of no value in dissecting L-LAC metabolism. The fluorescence searchmenu
microscopic studies by Lemire et al. [136] further revealed the localization of LDH within the
mitochondria. In that study, it was reported that “mitochondrial fractionation and western blot
analysis revealed that LDH1 isozyme populated predominantly the matrix/inner membrane
components. “Thus, in distinction with [79], we claim that the localization of the mitochondrial
L-LDH is no longer a matter of debate.
Question 4: Can cancer energy metabolism be described exhaustively with no
consideration for L-LAC transport and metabolism in mitochondria? The answer here is no
(and
 see above). 
We hope that all the sceptics can accept the above conclusion as any good student of
ours did!
Interestingly, in distinction with that reported by Brooks et al. [137] after 2014 when “The
mitochondrial L-lactate dehydrogenase affair” was published [22], to the best of our
knowledge, with the exception of Fulghum et al. [134], there was no denier of the existence of
the mitochondrial L-LDH.
Author Contributions
Conceptualization, draft preparation, writing, review and editing, S.P., A.S. and P.P. All
authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Acknowledgments
(/)
The authors are indebted to Ersilia, Maureen, and Gabriella for their silent, generous and
unconditional longstanding support.
searchmenu
Conflicts of Interest
The authors declare no conflict of interest.
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Passarella, S.; Schurr, A.; Portincasa, P. Mitochondrial Transport in Glycolysis and
Gluconeogenesis: Achievements and Perspectives. Int. J. Mol. Sci. 2021, 22, 12620.
https://doi.org/10.3390/ijms222312620
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Passarella S, Schurr A, Portincasa P. Mitochondrial Transport in Glycolysis and
Gluconeogenesis: Achievements and Perspectives. International Journal of Molecular
Sciences. 2021; 22(23):12620. https://doi.org/10.3390/ijms222312620
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Passarella, Salvatore, Avital Schurr, and Piero Portincasa. 2021. "Mitochondrial Transport in
Glycolysis and Gluconeogenesis: Achievements and Perspectives" International Journal of
Molecular Sciences 22, no. 23: 12620. https://doi.org/10.3390/ijms222312620
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Int. J. Mol. Sci. 2021, 22(23), 12620; https://doi.org/10.3390/ijms222312620

Submission received: 29 October 2021 / Revised: 16 November 2021 /

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12620-g011.png?1637965918) Versions Notes (/1422-0067/22/23/12620/notes)

2. How Mitochondrial Traffic in Energy Metabolism Is Easily and Quickly Investigated?

Table 1. Static approach to investigate the role of mitochondrial transport in energy

The existence of antiport processes is suggested by the swelling occurring only as a

transport can be deduced. Swelling inhibition due to a non-penetrant compound by itself

When using a metabolite/compound detecting system the reactions occurring outside

extramitochondrial phase, and measurements of mitochondrial ΔΨ generation. Having

Thus, to calculate ni, ne must be measured.

ni = 14Ccpm/SA − 3Hcpm/SAH2O × Ce − ViCe (under B condition).

3. Cytosolic NADH Oxidation via the Mitochondrial Shuttles

In glycolysis, NADH is formed in the reaction catalyzed by the glyceraldehyde-3-

Figure 5. Oxidation of extramitochondrial NADH by WKY5-RHLVM. Reconstruction of

Unloaded (A, B(a) and C) or mitochondria loaded with aminooxyacetate (AOA), an

4. Phosphoenolpyruvate (PEP) Transport in Mitochondria

9. The dependence of the rate of the pyruvate/[14C]-phosphoenolpyruvate (PEP)

5. L-Lactate Transport in Mitochondria

LAC/PYR antiporter and the oxidation of cytosolic NADH in a non-energy-competent L-

A better understanding of the role of L-LAC in neuronal energy metabolism and in

investigators interpreted as a mechanism of lactic acid clearance

- Question 1: Do L-LAC transport and metabolism occur in mitochondria? The answer

of mL-LDH in the zoom_out_map

This research received no external funding.

Institutional Review Board Statement

Informed Consent Statement

Data Availability Statement

The authors declare no conflict of interest.

2. Spinelli, J.B.; Haigis, M.C. The multifaceted contributions of mitochondria to cellular

64. Jin, (/)

Biophys. Res. Commun. 2015, 462,

e75154. [Google Scholar

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101177. [Google Scholar

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