Voi. 260,No. 1, Issue of January 10,pp. 292-299,1985
Printed in U.S.A.
THEJOURNALOF BIOLOGICAL
CHEMISTRY
0 1985 by The American Society of Biological Chemists, Inc.
Carrier-mediated Uptakeof Lactate in Rat Hepatocytes
EFFECTS OF pH AND POSSIBLE MECHANISMS FOR L-LACTATE TRANSPORT*
(Received for publication, January 17,1984)
Pierre Fafournoux, Christian Demigne, and Christian Remesy
From the Laborutoire des Mulodies M6taboliqws, Znstitut National de la Recherche Agronomiqw, Their, 63122 Ceyrut, France
The rate of uptake and the distribution
ratio between
intra-and extracellular compartmentsof L- and Dlactate were studied in hepatocyte preparations from
fed rats.L- and D-laCtate uptake apparently depended
on both passive diffusion and carrier-mediated components. The apparentK,,, of the high-affinity carrier
for L-lactatewas in the range of
1.8 mM. The reciprocal
of lactate sugcompetitive inhibitions between isomers
by
gestthat L- andD-lactatemightbetransported
distinct carriers. Lactate transport was inhibited by
various anions; pyruvate was the most potent anion,
whereasonlyhighconcentrationsofketonebodies
were effective. Acidic extracellular pH enhanced lactate uptake, this effect being more pronounced forLlactate. At low pH, L-lactate was concentrated into
hepatocytes, but its affinity for the carrier appeared
unchanged, suggesting the existence of a processgaining energy from thepH gradient across thecell membrane. In the hypothesis of a lactatem+ symport, the
affinity for H+ was not dependenton lactate concentration and the apparentX,,,
for H” correspondedto a pH
of 7.34. No trans-stimulation of lactate uptake after
prior loadingof the cells with pyruvate or lactate was
observed. The present data suggest
that, at physiological concentrations, lactate uptake by the liver might
be largely carrier-mediated and therate of transport
across theliver cell membrane may be of a magnitude
relatively comparable to the rate of metabolism.
The role of transport processes in the control of the hepatic
metabolism of glucogenic substrateshas been extensively
investigated on mitochondria for pyruvate entry (1)or exit of
dicarboxylic acids (2). In contrast,plasma-membrane carriers
have essentially been considered for amino acids and, recently,
for monocarboxylate such as branched-chain 2-keto acids (3).
In this view, although lactate is a major glucogenic substrate,
the mechanisms involved in lactate uptake are still
poorly
known. At physiological pH, lactate is almost entirely in the
ionized form, which should slowly moveacross the hydrophobic matrix of the membrane. It has actually been found
difficult to account for the observed balances of lactate across
the liver on the basis of concentration gradient between intraand extracellular compartments (4) or of simple ionic or
nonionic diffusion (5). The possibility that lactate uptake by
liver cells could be carrier-mediated had thus tobe considered,
since evidence of transporters for lactate has been provided
in plasma membrane from erythrocytes (6, 7) or Ehrlich
ascites tumor cells (8). Studies on perfused liver have shown
* The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked “aduertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
that D-lactate uptake could be carrier-mediated and stereospecific (9). In the same way, recent investigations (10)suggest that two components could beinvolved in L-lactate
transport in hepatocytes: a simple noncarrier-assisted diffusion, effective at high extracellular concentrations, and a
carrier which might mediate the major part of lactate uptake
at low external concentrations. The rise of cell pH consecutive
to lactate metabolism is in keeping with a transport asanionic
form (11,12),but theconcomitant process involved (lactate/
H‘ symport or exchange with OH- or other anions) is not
firmly established.
The underlying aim of these investigations is to assess
whether lactate transport could be rate-limiting, particularly
in situations of impaired hepatic utilization such as acidosis
(5)or high-protein diets (4).This requires precise evaluations
of the kinetic parameters of lactate transport, as well as, if
any, of associated ligands. Such investigations have probably
been slowed down bytechnical problems, since the time course
of lactate uptake has to be measured over very short periods
and in the absence of highly specific inhibitors of lactate
metabolism. Furthermore, as pointed out (IO),a considerable
diffusion component is observed in isolated hepatocytes,
which might be overestimated and irrelevant in uiuo. Using
an improved technique for measurement of the distribution
of lactate between intra- and extracellular compartments, we
have been able to show that thekinetic parameters for L- and
D-lactate transportaredistinctandthat
L-lactate uptake
might be essentially carrier-mediated in physiological conditions. The effects of changes in externalpHsupport the
hypothesis that anionic lactate could be transported with H‘,
with an apparent K , for H’ corresponding to physiologial
values. Furthermore, L-lactate transport was enhanced by
acidic pH,and even accumulated against a concentration
gradient in such conditions.
EXPERIMENTAL PROCEDURES
Prepuratwn of fsolnted Hepatocytes-Male Wistar rats weighing
approximately 250 g were housed in air-conditioned quarters with a
controlled 12-h light and dark cycle (dark cycle from08.00 to 20.00 h)
and received Sanders Laboratory Chow and drinking water ad libitum.
Hepatocytes were isolated from fedrats. Collagenase dissociation was
essentially performed as outlined by Berry and Friend (13) and Krebs
et ai. (14).Cells were then purified by centrifugation on a Percoll
gradient (15); the gradient was preformed by centrifugation of a 30%
Percoll solution in KRB’ buffer (pH 7.40,saturated with C02/02,
1:19, v/v) at 20,000 X g for 20 min in a fixed-angle rotor. The cell
suspension was layered on the top of the gradient and centrifugated
at 1,000 X g for 5 min in a swinging rotor. The pellets of viable cells
were resuspended in KRB buffer and centrifuged twice (200 X g, 1
min) to eliminate Percoll. Cells viability, estimated by cell-membrane
refractoriness in phase-contrast microscopy, ranged from 97 to 99%.
The incubations were performed in the KRB buffer containing 10
The abbreviations used are: KRB, Krebs-Ringer bicarbonate;
acid; DMO,
HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic
5,5-dimethyl-2,4-oxazolidinedione.
292
Lactate Transport in Hepatocytes
mM HEPES and 2% (w/v) albumin (fraction V) dialyzed against the
same medium.
Transport Experiments-Kinetic studies of entry of labeled lactate
require conditions minimizing the metabolism of intracellular lactate.
Aswillbe
discussed further,lactate metabolism could hardly be
blocked at the initial step of hepatic utilization, whereas effective
inhibitions of lactate metabolism are obtained with inhibitors of
transaminases (16) and of gluconeogenesis (17). In addition, incubations have been performed over very short periods. The cell suspension was thus preincubated for 5 min at 37"C in a KRB buffer
containing 1 mM aminooxyacetate and 1 mM 3-mercaptopicolinate.
Just before the experiment, the hepatocyte suspension was briefly
centrifuged (100 X g, 1 min) and resuspended (35 mg of cells/ml) in
fresh KRB buffer containing the inhibitors to eliminate the remaining
amount of lactate, essentially originating from glycogenolysis.
Lactate transport was initiated in Eppendorf Microfuge tubes
containing L- or D-('4C]lactate and ['Hlinulin in 100 pl of KRB buffer
by addition of 300 pl of hepatocytes suspension. The resulting BUSpension was immediately mixed and incubated at 37 "C for 10 S. For
measurements of the time course of lactate uptake, 1.5 ml of hepatocyte suspension were incubated at 37 "Cwith labeled L- or D-laCtate
and inulin, with appropriate agitation; samples (300 p l ) of the suspension were taken at the required time. For both procedures, cells
were immediately separated from the medium (within about 2 8) by
rapid centrifugation (Boo0 X g, 10 8 ) through 250 p1 of silicon oil (AR
200/AR 20,1:3, v/v) layered on the topof 50 pl of 1.2 M HCIO,. After
the spin, 100 pl of the supernatant were transferred to counting
minivials and mixed with 3 ml of scintillation liquid (Insta-Gel). The
remaining supernatant and about half of the oil layer were then
discarded by vacuum aspiration, the microtube was plunged in liquid
N2 (about 5 s), and the bottom (containing the cell pellets in frozen
perchloric acid) was immediately cut about 3 mm above the lower
meniscus of the oil layer and transferred into a counting minivial.
250 ~1 of distilled water were added as were after vigorous agitation,
3 ml of scintillation liquid. It was checked that thisprocedure allows
a quantitative recovery of label in cell pellets, without contamination
by the medium on the top of the oil layer. The correction for
extracellular fluid was in the range of 35% of centrifuged water.
For very short kinetic studies OS), the above procedure has
been modified to minimize the time elapsing between addition of the
cells to theincubation medium and separation by centrifugation. For
this purpose, 500 pl of incubation medium containing labeled lactate
and inulin were put on the top of the oil layer in microfuge tubes.
The tubes were equilibrated at 37 "C and transferred in a microfuge
rotor 5 s before the onset of incubation. This was initiated by addition
of 50 p1 of hepatocytes suspension (60 mg/ml), homogenization being
performed by rapid repipetting of the medium. At the required time,
the cells were separated from the medium by rapid centrifugation
(8000 X g, 10 8 ) . With this procedure, the limit for reproducible
measurements of lactate entry was decreased to about 3 s.
Evaluation of Lactate Metabolism during Kinetic Studies-Complete oxidation into CO, or utilization for glucose production was
assessed inseparateexperiments in which hepatocytes (35mgof
cells/ml, 3 ml) were incubated with ~-[U-"C]lactate(2 pCi) over 10,
20, and 60 s in the presence or absence of the metabolic inhibitors.
The reaction was stopped by injection of 1 ml of 1.2 N HClO4 into
the capped flasks, 0.2 ml of Hyamine was added into the center well,
and COa wascollected during a 1-h incubation at 37 "C with shaking.
Pdioactive incorporation into glucose was determined on the neutralized perchloric extract after separation from lactic acid on a
Dowex 1-X8 (100-200 mesh) column (18), then purification as glucose
pentaacetate (19). The recovery of label as lactate during short-term
incubations was assessed on the perchloric extracts of the cell pellets
after centrifugation through silicon oil (see above). Aliquots were
either sampled for total 14Cmeasurement or submitted to procedure
for purification of ("C]lactate. For this purpose, the acidic extract
was first poured into microfuge tubes containing 300 pl of hydrazide
gel to trappyruvic acid (and possibly other keto acids). The hydrazide
gelwas prepared by reacting hydrazine hydrate on polyacrylamide
beads (Bio-Gel P-60, 100-200 mesh) as described by Hayashi et al.
(20). The supernatant and the wash fractions were pooled, neutralized, and passed through a Dowex 1-X8 resin column. After washing
with deionized water, acids were eluted with 5 ml of 1 N formic acid
and counted for radioactivity. Contamination of this fraction by acidic
compounds, apart from pyruvic acid, was checked on separate neutralized aliquot8 by chromatography on Whatman No. 1 paper. The
separation was carried out in a saturated tank using 1-butanol/acetic
293
acid/H20 (12:3:5), followedby air drying. One-centimeter strips were
cut and counted in scintillation vials. The purity of the lactate spot
was determined from parallel assays in which samples were pretreated
with L-lactate dehydrogenase (21). This procedure showed that label
in organic acids submitted to resin purification was essentially (98%)
in lactic acid, and the procedure of paper chromatography was not
utilized further.
Measurements of Aqueous CeU Volume-This was determined by
incubating the hepatocytes in media with 'H20 and ["C]polyethylene
glycol for 5 min at 37 "C.Measured values were 0.45 f 0.04 pl/mg of
cells. The distribution ratio is referred to as theratio of intracellular
to extracellular concentrations.
Determination of Intracellular pH-The distribution of [14C]DM0
across the inulin-impermeable membrane was used (22); [aH]inulin
(50 pg/ml) was added together with 0.5 mM [14C]DM0, and the
distribution of labeled compounds across the membrane waa measured after a10-min incubation (23). The external pHwas determined
at 37 "C with an Orion 701 pH meter, and the apparent intrace1l:dar
pH was taken as:
Apparent pHi = pK
+
where [DMO]. and [DMO]i represent the sum of the concentrations
of the charged and uncharged forms of DM0 in the extracellular and
intracellular compartments, respectively, and pK was taken at 6.13
at 37 "C.
Chemicals-~-[U-"C]Lactic acid (160 mCi/mmol), D-[U-"C]lactic
acid (40 mCi/mmol), ['Hlinulin (1.20 Ci/mmol), and ['4C]polyethylene glycol (22 mCi/g) were purchased from The Radiochemical Centre
(Amersham, Bucks, United Kingdom); 3H20 (1 mCi/ml) and ["C]
DM0 (54 mCi/mmol) were obtained from the Commissariat a
I'EnergieAtomique (Gif-sur-Yvette, France). Unlabeled DMO, Llactic acid, D-laCtiC acid, albumin (fraction V), and aminooxyacetate
were from Sigma. Collagenase was obtained from Boehringer (MeyIan, France), Percoll from Pharmacia Fine Chemicals (Uppsala, Sweden), silicon oils from Wacker Chemie (Munich, Germany), and
Dowex 1-X8 resin and Bio-Gel P-60 polyacrylamide gel from BioRad. 3-Mercaptopicolinate was a kind gift from Dr. Di Tullio, Smith
Kline & French (Philadelphia, PA).
RESULTS
Measurement of lactate utilizationshowed that, in the most
exacting conditions (external L-lactateat 0.2 mM), no significant label was recovered in glucose up to60 a, when inhibitors
were present in the medium. Appearance of radioactivity in
COn was not detected up to 10
s and amounted to1.5% of 14C
uptake after 60 a. When inhibitors were omitted, 14C0,production was three times higher, but incorporation
of label into
glucose was barely detectable. The percentage of "C in CO,
or glucose remained below 1%with external lactate higher
than 0.6 mM. Besides completeoxidation andgluconeogenesis,
interferences might have arisen from incorporation of radioactivity into various intracellular metabolites such as pyruvate or various products of its subsequent metabolism. Measurement of the recovery of label as lactate in cell pellets
showed that lactate accountedfor 97% of label after 10 s and
94% after 30 s a t 0.2 mM external lactate in the presence of
inhibitors. The main contamination was identified as keto
acids trapped by the hydrazide gel, probably pyruvate, which
accounted for 2% (10-s incubation) and 3% (30-9 incubation)
of the label. Thus, it maybe assumed that there was practically no interference from metabolism during short-term kinetic studies of lactate entry.
Time Course of Lactate Influx in Hepatocytes-Fig. 1 shows
the uptake of L- or D-lactate at 0.2 mM each in the medium
over a 2-min period, and the insetshows the detailed kinetics
of L-lactate entry over the first 12 s of incubation. The rate
of entry was faster for L-lactate, but readily declined with
time; asa result, a steady-state level was practically attained
within 1min. Conditions of initial rateof L-lactate entrywere
observed over a t least 12 s; however, the regression line did
Lactate Transport in Hepatocytes
r
I
\
l
I
30
Go
I
90
Time (sec)
FIG.1. Time course of lactate uptake and accumulation into
liver cells.Hepatocytes were suspended in KRB medium containing
0.2 mM L- or D-lactate at 37 "C and then, at the required time,
aliquots of the incubation mediumweretransferred into separate
microfuge tubes for separation across a silicon oil layer. The calculation of the distribution ratio was based on the intracellular water
volume of 0.45 pl/mg of cells. In the inset is shown the detailed time
course of lactate uptake; the above procedure was modified in that
incubation, and separation of the cells across the oil layer was carried
out in the same microfuge tube (see "ExperimentalProcedures").The
results shown are the averages ? S.E. of duplicate determinations for
two batches of cells.
not intercept with the y axis at the origin. This may reflect
various factors: unspecific fixation of label on hepatocytes,
underestimation of extracellular medium trapped with cell
pellets. This interference was systematically subtracted in
further studies, but it was limited (cO.01 nmol/mg of cell at
0.2 mM) and was independent of lactate concentrations. The
distribution ratio at 0.2 mM external lactate, measured after
2 min, was about 0.8 for L-lactate against 0.4 for D-lactate.
The determination of this ratio may be somewhat inaccurate
owing to the difficulty of complete inhibition of lactate metabolism. The time course of entry has also been examined
for higher concentrations of L-lactate (1 and 30mM); the
distribution ratio was not noticeably modified, whereas the
duration of the initial period of high rate of entry was extended.
Kinetic Analysis of Lactate Transport-Changes in extracellular lactate affected the uptake of labeled lactate; the
entry rate was enhanced with increasing concentrations in
the medium, but reached a plateau at extraphysiological values. Fig. 2 shows the relation between velocity of total lactate
uptake and the extracellular concentration. The LineweaverBurk plot (inset) deviated from linearity for high values of s
(> 5 mM) and intercepted with the origin, which is generally
interpreted as passive diffusion. Extrapolation of the experimental plot for lactate concentrations lower than 2 mM suggests that the kinetic parameters of the component having
the highest affinity are different for L- and D-laCtate. The
apparent K,,, was about 1.9 mM for L-lactate, in the range of
FIG.2. Kinetics of lactate transport by rat hepatocytes
(Woolf-Augustinson-Hofstee representation. The velocity of
lactate transport was measured at various extracellular concentrations between 0.2 and 30 mM. The lactate uptake was measured for
10 s at 37 "C.Inset, Lineweaver-Burk representation.
physiological values, but was markedly higher for D-laCtate,
at 4.5 mM. The VmaXvalues were similar for L- and D-lactate
transport: about 0.6 nmol-mg" of cells. 10 s-'. The plot of u
against u/s was curvilinear with both isomers; this may fit a
theoretical model describing uptake by low- and high-affinity
pathways for each. It appears that, with this representation,
only L-lactate transport parameterscould betentatively quantified. One of the components is a relatively high-affinity
process with an apparent K, of about 1.8 mM and a Vmaxof
0.6 nmol. mg" of cells. 10 s-'. These values are in agreement
with data yielded by the Lineweaver-Burk plot. The evaluation of the parameters of the low-affinity component is more
difficult, owing to its very high apparent capacity and its low
affinity (apparent K,,, in the range of 30-40 mM). In fact, its
specificity and its relevance in physiological conditions may
be questioned.
Effects of Inhibitors-In order to further characterize lactate transport, theeffects of various anions and alaninehave
been tested (Table I). SO$- failed to inhibit lactate transport,
suggesting that the general anion transporter is not directly
involved in lactate uptake. Most of the physiological anions
examined inhibited the uptake of lactate (0.4 mM in the
medium), some of them to a larger extent than lactate itself.
Pyruvate appeared as the most potent inhibitor of lactate
tranport (both L- and D-isomers). Ketone bodies provedto be
efficient inhibitors of L-lactate uptake, but acetoacetate displayed a more pronounced effect on D-lactate transport than
did 3-hydroxybutyrate. Volatile fatty acids, which are extensively removedby the liver, also decreased the transportof Dand L-lactate, propionate being more efficient than acetate.
Alanine did not affect lactate entry. t-Lactate transport was
weakly inhibited by D-lactate, whereas D-lactate transport
was markedly inhibited by L-lactate.
Analysis of these processes by Dixon plot showed that L-
Lactate Transport in Hepatocytes
295
TABLEI
Inhibition of L- and D - h t a t e transport by various anions and
alanine
The uptake of 0.4 mM ["Cllactate by hepatocytes in suspension
was determined inthe presence of the KRB buffer orof the indicated
inhibitors at 10 mM. A velocity of 0.22 nmol. mg" of cells. 10 s-' (Llactate) and of 0.08 nmol. mg" of cells. 10 s-l (D-lactate) was found
in the absence of inhibitor. The results are the average S.E. of
three determinations for two batches of cells.
external p H (Fig. 4A),these changes being quite significant
in the range
of physiological values. The transportof D-lactate
appeared less responsive to pHvariations. The plot of lactate
entry against ApH indicates that this rate increasing
is
when
ApH is reduced or even reaches negative values (Fig. 4B). As
a result, the correlation between ApH and entry rate was
apparently linear. Since variations in intracellular pH appear
attenuated compared to external pH, limited changes
ApHin
L-Lactate
D-LaCtate
may
give
rise
to
substantial
alteration
of
the
rate
of
L-lactate
Inhibitor
transuort
transDort
uptake.
% inhibition
The strikingrise in lactate uptakewith acidic p H elicited a
11.2 f 1.2 16.3 f 1.1
Na2S04
substantial accumulation of L-lactate in hepatocytes, with a
15.6 f 0.5 10.7 f 1
Alanine
distribution ratio of 1.9 for p H 6.40 in the medium (Fig. 5).
72 f 3
72 f 4
Pyruvate
In very acidic and unphysiological conditions (pH 5.33), the
41 f 2
45 f 3
Acetoacetate
ratio even reached a value of about 3 (result not shown). This
42 f 3
32 f 2
Acetate
53 t 6
46 f 5
Propionate
accumulation suggests the existence of accompanying mech28 f 3
19 f 2
DL-&Hydroxybutyrate (10 mM)
anisms which would provide the required energy, such as pH
34 f 2.5
DL-P-Hydroxybutyrate (20 mM)
45 f 2
gradient.
45 f 4
38 k 6
L-Lactate
Processes Involved in Lactate Transport-The kinetics of
18f 1
28 3
D-LaCtate
lactateuptake was examined forvarious pH,inorderto
determine if affinity for lactate was affected by changes in
[H']. A plot of l / v against l/s demonstrates that the apparent
K,,, for lactate is not affected by p H (Fig. 6A). In the same
way, the plot of l / v against 1/[H+] hasbeen also drawn (Fig.
6B), yielding anapparent K,,, of 4.54 X
(corresponding
t o a pH of 7.34).
In order to assess whether lactate entry
closely
is related to
the external concentration of the dissociated or protonated
form,therate
of L-lactatetransport was plottedagainst
calculated values of undissociatedlacticacid
(Fig. 7). The
variation of the undissociated form was obtained by manipulating externalpH (at1 mM lactate) or lactate concentrations
at pH 7.4. The rate of lactate uptake was strikingly higher
when changes in the undissociated form corresponded to an
increase in total lactate. This indicates that theof rate
lactate
transport is not a simple function of external undissociated
lactic acid, but could be more closely related to the concentration of the anionic form. Theoretically, if the carrier were
specific for the dissociatedformalone,
the rate of uptake
should decrease with acidic pH. Theexplanation for the
increase is most likely that a proton(s) is also a substrate for
6
the
reaction, as supported
by other data,
5
6
7
Extracellular pH (ptia)
If the transport of lactate were to require the simultaneous
FIG. 3. Changes in apparent intracellular pH and in pH inward transport of H+, a relationship should exist betweeq
gradient across the cell membrane (pH, - pHi): difference in the distribution of lactate across the cell membrane in the
response to modifications of extracellular pH. The pH of the steady state and the pH gradient in these
conditions. In a
cell suspension was adjusted by the addition of small volumes of 2 M
steady
state,
the
rates
of
inward
and
outward
transport are
NaOH or HCl. The cells were incubated 10 min with 0.5 mM ["C]
D M 0 and 50 pg/ml [3H]inulin.The reaction was stopped by centri- equal, and it follows that: [lactate],. [H']: = [lactateli. [H+]:
fuging the cell suspension through a layer of silicon oil as described and,
under "Experimental Procedures." Immediatelyafter centrifugation,
pH of supernatant was measured at 37 "C. The results are means
log [lactate]; = 4PHi - PHJ
S.E. of three determinationsfor the same batch of cells.
*
~~~
~
*
1
*
and D-lactate exert reciprocal inhibition on their transport.
In both cases, the apparent Ki for inhibition of the transport
of the opposite isomer was largely higher than the K,,, for
uptake; for example, concerning L-lactate, the K, for inhibition of D-lactate transport was 40 mM against 4.5 mM for the
K, for D-lactate transport. In the sameway, inhibition of Llactate uptake by pyruvate was also competitive, but its Ki
was relatively low (about 3.5 mM).
Effect of pH-Lactate transport
may also be affected by
changes in extracellular pH, which elicits striking modifications of the ApH between cells and medium (Fig. 3). The rate
of lactate uptake was almost linearly enhancedby decreasing
(=)
where the subscriptsi and e represent intra- and extracellular
concentrations, respectively. In Fig. 8, it appears that there
was alinear relationshipbetween log( [la~tate]~/[lactate].) and
the pH difference. Taking the values for the slope and the
intercept, it comes that x is equal to 1.08. These data are
consistent with a mechanism in which entry of L-lactate is
accompanied by the inward transport of approximately 1 H'
ion. The fact that the intercept with the ordinate is close to
0 indicates that no additional term is required in the above
equation. However, it must be kept in mind that the determination of the distribution ratio may be affected by incomplete inhibition of lactate metabolism.
Lactate Transport in Hepatocytes
296
FIG. 4. Influence of pH on L- or Dlactate uptakeby rat hepatocytes. A,
the velocity of lactate transport was plotted against the external pH. B, the velocity of lactate transport was plotted
against the pH gradient through the cell
membrane.
'
\
DISOMER
I
-10
1
-0.5
I
0
I
+os
external pH -internal pH
"
.-b
+2
pH 7'45
.
5
0
50
100
150
200
(sec)
Time
FIG. 5. Influence of pH on the time course of L-lactate transport and on the distribution ratio. The uptake of 1 mM L-["C]
lactate was measured at pH 6.40, 6.80, and 7.45. The distribution
ratio is indicated. The results shown are the averageof duplicate
determinations from two different hepatocytes suspensions.
DISCUSSION
Methodological Conditions-A major difficulty encountered
in studies dealing with carriers lies in interferences from
metabolism of transported substrates. Hepatocytes from fed
rats may, as far as glycogen stores are present, produce large
amounts of lactate. As a result, intracellular lactate increases
with subsequent alteration of specific radioactivity. However,
the various steps of isolation of hepatocytes (purification of
viable cells, removal of Percoll, preincubation) afford periods
of glycogenolysis for about 1 h. After the cells have been
resuspended in fresh medium, lactate concentration was very
) remained practically unchanged throughout
low ( 6 0p ~and
the incubation. Lactate, particularly the L-isomer, may be
readily metabolized, and its label may enter in numerous
metabolites. Addition of aminooxyacetate and 3-mercaptopicolinate might be inadequate for complete inhibition of
lactate metabolism. Oxamate, or N-substitutes derivatives,
may constitute potent inhibitors
of lactate dehydrogenase
(24). However, such compounds have to be utilized at very
high concentrations and exhibit noticeable side effects (25);
in addition, such inhibitors could not be used for comparison
of L- and D-lactate transport, since D-lactate metabolism bypasses the lactic dehydrogenase step. Transaminase inhibitors
have been shown to efficiently block lactate utilization (16)
by various means: rise of the cytosolic NADH/NAD ratio (26)
and disturbances of the malate-aspartate shuttle (27), hence
indirectly affecting the activity of pyruvate dehydrogenase.
Accordingly, under our conditions (10-s incubations), lactate
metabolism appeared extremely limited; this is all the more
relevant since hepatocytes from fed rats slowly metabolize
lactate.
Churacteristics of Lactate Transport-Previous investigations supported the view that lactate uptake is mediated by
two components in hepatocytes: a carrier-mediated system
and a nonsaturable process (10). With more detailed kinetics,
our data allow us to distinguish different components for
lactate transport. The low-affinity component could be tentatively attributed
to
passive diffusion for D-lactate, since the
Woolf-Augustinson plot yielded a relatively vertical assmptote at high concentrations. Athigh concentrations of Llactate, besides passive diffusion itself, a low-affinity carrier
could also be involved,but possibly with a poor specificity for
lactate transport. For the high-affinity component, kinetic
parameters appear different for the L- or D-isomer, affinity
being higher for L-lactate ( K , = 1.8 mM against about 4.5 mM
for D-lactate), whereas transfer capacity is practically similar
for both isomers (about 0.6 nmol .mg"of cells-10 s-'). In
fact, the present data do not necessarily reflect the existence
of several saturable systems, since such biphasic kinetics are
displayed by a similar system, the lactose/H+ cotransport
(28), which is composed of a single polypeptide.
The question arises as to whether lactate could be transferred by the general anion carrier. The failure of SO:- to
inhibit L- or D-lactate transport supports the
view that a more
specific component is involved in lactate uptake. The existence of a nonspecific anion carrier in the liver has even been
questioned (29); in fact, Brachtet al. (30) showed that a
carrier for inorganic anions (particularly SO:-)does exist in
the plasma membrane of the liver, but this carrier is independent of the monocarboxylate carrier, although it shares
some common inhibitors.
Each isomer of lactate competitively inhibits the transport
Lactate Transport in Hepatocytes
297
i
FIG. 6. Kinetic analysisof the inflaence of pH on L-lactatetransport. A, the velocity of lactate transport
was plotted against lactate concentrationsaccording to theLineweaver-Burk representation at pH 7.45,7.05, and
6.05. B, the velocity of lactate transport was plotted against H+ concentrations according to theLineweaver-Burk
representation at lactate concentrations of 0.6, 1.5,5, 15, and 30 mhp.
4
I-
0
2
4
[lACTC
6
8
A C i a <pM)
FIG. 7. Reiation between external undissociated form of Llactic acidand the rateof transport. Variations of undissociated
form were obtained by two ways: constant pH (7.4) and increasing
concentration of total lactate (0.4,0.8, 1, 1.5,3, 10,20, and 30 m ~ ) ;
constant concentration of lactate (1 mM) and with changes in external
p H (8.30, 7.67,7.46, 6.97,6.48, and 6.03). b s u l t s are the average of
two deCerminations fromthe same batch of cells.
FIG. 8. Steady-state distribution of L-lactate and B'. The
pH of the cell suspension was adjusted at 8.30, 7.40, 7.17, and 6.77.
L-tactate distribution was measured by the means of three
of theoppositeisomer,but
the mostpotentinhibition is The
determinations when the accumulation was plateauing. The DM0
exerted by L-lactate on D-lactate transport.
Since the I%.values distribution was determined in parallel incubations (10 min). The
are markedly different fromthe K , values, it may be assumed results are the average of three determinations from two batches of
that separate carriers are operating
for each isomer. However, cells.
298
TransportLactate
the physiological relevance of such reciprocal inhibition is
questionable, since the highest concentrations of the L- or Disomers observed in vivo could hardly affect lactate-transport.
It is noteworthy that pyruvate appeared as the most potent
inhibitor for transport of both isomers. Since pyruvate exhibits a high affinity for the lactate carrier, this raises the
possibility that pyruvate could also be transported by this
carrier, or possibly exchanged with lactate. In fact, in ratsfed
high-carbohydrate diets, opposite fluxes of pyruvate and lactate across the liver havebeen observed in vivo (4). Nevertheless, the possibility of a lactate/pyruvate exchange is not
consistent with the lack of trans-stimulation of lactate transport after preloading of the cells with pyruvate (results not
shown); furthermore, the physiological concentrations of pyruvate in the liver would be too low for a significant effect on
lactate transport. In contrast, in rabbit erythrocyte, which
has a high capacity for outward transport of lactate, lactate/
lactate and lactatelpyruvateexchanges are considerably more
rapid than lactate/H+ cotransport (31). The effect of ketone
bodies was alsointeresting to consider since these anions are
released in large amounts during starvation and could be a
candidate as ligand for a lactate/intracellular anion antiport,
in the same way as accelerated exhange of cytosolic pyruvate
for the intramitochondrial acetoacetate on the monocarboxylate translocator (32). In fact, ketone bodies affected lactate
transport, but at concentrations corresponding to severe hyperketonemia. These interactionsnevertheless require further
investigations; it must be noted that, in vivo, maximal efficiency of lactate uptake is frequently concomitant to situations of active ketogenesis.
Propionate and alanine are
the major glucogenicsubstrates
removed by the liver in rats fed standard diets(33),and they
have been reported to inhibit lactate utilization in certain
conditions (4, 34). It appears that the inhibition of lactate
transport displayed by propionate is notsignificant for physiological concentrations in portal blood (<0.5 mM). L-Alanine
was inefficient; recent observations (35) have demonstrated
that L-alanine stimulated the binding of ['4Cjlactate to plasma
membrane, but without obvious effects on lactate transport
itself.
Effects of pH on Mechanisms of Lactate Transport-Acidic
pH markedly enhanced lactate transport; this fact could be
ascribed to an increase in the percentage of the non-ionic
form or to changes in the H" gradient between both sides of
the cell membrane. Variations in theundissociated form were
obtained by manipulating pH or the total concentration of
lactate in the medium for a fixed pH; assuming that undissociated lactate were the only permeant form, similar results
should be obtained in both conditions. In fact, lactate uptake
appeared markedly higher when total concentration of lactate
was elevated; this is in line with a simultaneous entry of the
anionic form. The undissociated form can actually diffuse
across the cell membrane, but the concentration of this form
is extremely low at pH 7.2-7.4; however, diffusion of the
undissociated lactic acid might partly account for accelerated
uptake with very acidic pH. These points support the view
that the flux of anionic lactate represents the major part of
lactate uptake by the liver.
The specific roleof the gradient of pH hasbeen investigated
since the carrier-mediated transport of lactate may correspond to a H+ symport or a OH- antiport. The determination
of cell pH by DM0 repartition showed that the drop of cell
pH was relatively dampened, compared to external pH, so
that the ApH between both sides of the cell membrane was
depressed by acidosis. However,it must be kept in mind that
the present values reflect the resultant of both cytosolic and
in Hepatocytes
mitochondrial compartments (36); as a result, the actualApH
might be slightly underestimated. Modifications of pH are
likely to influence the rate of lactate transport by affecting
the H' and OH- gradients. It is noteworthy that hepatocytes
accumulated L-lactate against a concentration gradient (about
2-fold) with the most acidic pH. Despite the fact that there
are similar correlations between the rateof lactate entry and
the ~ a ~ e noftN"
s and OH-, the accelerated uptake of lactate
concomitant to a drop of cell OH- during acidosis would not
fit with the existence of a lactate/OH- antiport, particularly
if the apparent i(,for OH- was, as calculated for H+, close to
physiological values. Onthe hypothesis of a lactate/H+ symport, probably operating by a mechanism of random fixation
of lactate andH+ andwith apparent K, for both ligands very
close to values reported in the portal vein, a preliminary
model maybe proposed in which the influx of one lactate ion
is accompanied by the inward entry of one H+ ion. The most
classic system of substrate/H+ symport has been described
for lactose transport in Escherichia coli, the relevance of this
model being established from various approaches besides kinetic studies of uptake rate of &-galactosideas a function of
pH (37). A similar H' movement coupledto lactate transport
in Escherichia coli has alsobeendescribed
(38). In such
models, a substrate gradient drives H+ entry and,conversely,
H' or electrochemical gradients drive the accumulation of
substrate. In hepatocytes, the more acidic pH of the cytosol
should prevent such a system to drive lactate entry,except in
very acidic conditions when cytosolicpH become higher than
external pH. In contrastto the electrogenic transport of
lactose, it is difficult to distinguish between a system catalyzing the symport of a H' and a lactate anion and one catalyzing
the uniport of lactic acid. The only difference is that in the
uniport the proton is bound to the lactate carboxyl, whereas
in the symport it is bound elsewhere on the carrier molecule.
The present data support the hypothesis of a carrier for the
anionic form of lactate, but an alternativemodel in which the
carrier would be highly specific for the protonated form and
affected by changes in pH cannot be ruled out. However, it
must be kept in mind that the distribution ratio of total
lactate we measured in physiological conditions corresponds
to a ratio of 1.8 for lactic acid; as a result, in the hypothesis
of a carrier for the protonated form, the question of energization of such a process arises. In mammalian, it must be
noted that H" fluxes induced by lactate gradients have only
been demonstrated on particular models (rabbit erythrocyte,
tumor cells) which havean especially highcapacity for lactate
transport (8,31).The lower sensibility of D-lactate transport
to pH changes strengthens theview that itsentry is mediated
by a distinct system, but the question as to the rise of the
protonated form which could
account for the enhanced uptake
with acidosis requires further investigations.
Physiological Consequences-The contribution of the diffusion component, if any, is probably much lowerin vivo than
in isolated hepatocytes in relation to the exposure of the
totality of the cell membrane to extracellular lactate and,
possibly, the loss of metabolic zonation. Nevertheless, even
in vitro, for physiological concentrations (1-5 mM), the contribution of the carrier-mediated process for lactate uptake
could be in therange of 80%for 1 mM L-lactate, declining to
about 60% at 5 mM. The physiolo~ca~
concentrations of Dlactate are usually very low in the portal vein; as a result, Dlactate uptake might be essentially carrier-mediated in the
liver. Comparison of the rate of transport with metabolic
fluxes observed in vivo, for lactate concentrations in the range
of 1 mM, indicates that both processes could be of relatively
comparable magnitude (1.6 gmol of lactate transported per
Lactate Transport in Hepatocytes
299
min/g of cells against about 1 pmol metabolized per min/g of 12. Baron, P. G., Iles, R. A., and Cohen, R. D. (1978) Clin. Sci. Mol.
Med. 55,171-181
liver), differing from data of Monson et al. (10) who suggeited
13. Berry, M. N., and Friend, N. W. (1969) J. Cell Biol. 43,506-520
that the initial rate of entry could be six times higher than 14. Krebs, H. A., Cornell, N. W., Lund, P., and Hems, R. (1974) in
the measured rate of metabolism at 1.9 mM. Our data support
Regulutwn of Hepatic Metabolism (Lundquist, F., and Tygstrup,
the view that, under some physiological disturbances, lactate
N., eds) pp. 726-750, Munksgaard, Copenhagen
transport might become rate-limiting. However, the rate of 15. Dalet. C.. Fehlmann., M.., and Debev.
- . P. (1982)
.
. Anal. Biochem.
122,119-123
lactate utilization in vivo is obtained in the presence of very
limited gradients of concentration between afferent blood and 16. Taylor, W. M., Reinhart, P. H., and Bygrave,F. L. (1983)
Biochem. J. 212,555-565
liver, whereas the concentration gradients in vitro between 17. Goodman, M. N. (1975) Biochem. J. 150,137-139
intraand
extracellular compartments
are
considerably 18. Woods, C.L., Babcock, C. J., and Blum, J. J. (1981) Arch.
higher. The stimulation of lactate uptake in hepatocytes with
Biochem. Bwphys. 212,43-53
acidic external pH constitutes an interesting feature, suggest- 19. Jones, G. B. (1965) Anal. Biochem. 1 2 , 249-258
ing that the blockade of lactate utilization during acidosis 20. Hayashi, T., Tsuchiya, H., Todoriki, H., and Naruse, H.(1982)
Anal. Bwchem. 1 2 2 , 173-179
(39) is restricted to the intracellular steps of lactate metabo- 21. Brandt, R.B., Waters, M. G., Rispler, M. J., and Kline, E. S.
lism. In the same view, it could be interesting to assess the
(1984) Proc. SOC.
Exp. Biol. Med. 175,328-335
role of transport processes in the phenomenon of lactate 22. Roos, A., and Boron, W. F. (1981) Physwl. Rev. 6 1 , 296-334
release observed in rats fed high-protein diets, in the absence 23. McGivan, J. D. (1979) Biochem. J. 182,697-705
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clue to evaluate the actual importanceof transport for lactate
Bwl. Chem. 240,2191-2195
metabolism in the liver.
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