(Received for publication, June 13, 1995; and in revised form, October 24, 1995)
From the
The kinetics of transport of L-lactate, pyruvate,
ketone bodies, and other monocarboxylates into isolated hepatocytes
from starved rats were measured at 25 °C using the intracellular
pH-sensitive dye, 2`,7`-bis(carboxyethyl)-5(6)-carboxyfluorescein, to
detect the associated proton influx. Transport kinetics were similar,
but not identical, to those determined using the same technique for the
monocarboxylate transporter (MCT) of Ehrlich Lettré tumor cells (MCT1) (Carpenter, L., and Halestrap, A. P.(1994) Biochem. J. 304, 751-760). K values for L-lactate (4.7 mM), D-lactate (27 mM), D,L-2-hydroxybutyrate
(3.3 mM), L-3-hydroxybutyrate (12.7 mM), and
acetoacetate (6.1 mM) were very similar in both cell types,
whereas in hepatocytes the K
values were
higher than MCT1 for pyruvate (1.3 mM, cf. 0.72
mM), D-3-hydroxybutyrate (24.7 mM, cf. 10.1 mM), L-2-chloropropionate (1.3
mM, cf. 0.8 mM), 4-hydroxybutyrate (18.1
mM, cf. 7.7 mM), and acetate (5.4
mM, cf. 3.7 mM). In contrast, the hepatocyte
carrier had lower K
values than MCT1 for
glycolate, chloroacetate, dichloroacetate, and
2-hydroxy-2-methylpropionate. Differences in stereoselectivity were
also detected; both carriers showed a lower K
for L-lactate than D-lactate, while
hepatocyte MCT exhibited a lower K
for D- than L-2-chloropropionate and for L- than D-3-hydroxybutyrate; this is not the case for MCT1. A range of
inhibitors of MCT1, including
-cyanocinnamate derivatives,
phloretin, and niflumic acid, inhibited hepatocyte MCT with K
values significantly higher than for tumor
cell MCT1, while stilbene disulfonate derivatives and p-chloromercuribenzene sulfonate had similar K
values in both cell types. The branched chain
ketoacids
-ketoisocaproate and
-ketoisovalerate were also
potent inhibitors of hepatocyte MCT with K
values of 270 and 340 µM, respectively. The
activation energy of L-lactate transport into hepatocytes was
58 kJ mol
, and measured rates of transport at 37
°C were considerably greater than those required for maximal rates
of gluconeogenesis. The properties of the hepatocyte monocarboxylate
transporter are consistent with the presence of a distinct isoform of
MCT in liver cells as suggested by the cloning and sequencing of MCT2
from hamster liver (Garcia, C. K., Brown, M. S., Pathak, R. K., and
Goldstein, J. L.(1995) J. Biol. Chem. 270, 1843-1849).
Most mammalian cells transport lactic acid across their plasma
membranes by means of a monocarboxylate/proton cotransporter (MCT), ()the characteristics of which have been investigated in a
variety of cell types (see Poole and Halestrap(1993)). The most
extensively studied MCT is that found in the red blood cell (Deuticke,
1982; Poole and Halestrap, 1993), which has been identified and
partially purified in this laboratory (Poole and Halestrap, 1992).
N-terminal sequencing (Poole and Halestrap, 1994) has shown it to be
the same as the transporter recently cloned and sequenced from Chinese
hamster ovary cells and named MCT1 (Garcia et al., 1994).
Detailed studies of the kinetics and substrate and inhibitor
specificity of monocarboxylate transport into heart cells has indicated
that this tissue contains two isoforms of MCT that each have properties
distinct from MCT1 (Poole et al., 1989, 1990; Wang et
al., 1993, 1994, 1995). Immunofluorescent studies using
MCT1-specific antibodies have shown that heart cells also contain a
small amount of MCT1, but this is confined to the intercalated disk
region (Garcia et al., 1994). Data from other laboratories
suggest that the transporter found in skeletal muscle is yet another
isoform (see Roth(1991) and Poole and Halestrap(1993)), and
immunofluorescent studies of white muscle support these conclusions
(Garcia et al., 1994).
The hepatocyte requires a very rapid
transport mechanism for the uptake of lactic acid for gluconeogenesis
and lipogenesis while lactic acid efflux occurs under hypoxic
conditions. In addition, the liver is the major producer of ketone
bodies and exports acetoacetic acid and -hydroxybutyric acid
during fasting or endurance exercise (see Poole and Halestrap(1993)).
Lactate transport into liver cells has been investigated in this and
other laboratories using radioactive tracer techniques (see Poole and
Halestrap(1993)). Although some studies have been performed in vivo (Lupo et al., 1990), in the perfused liver (Schwab et
al., 1979; Bracht et al., 1981) and in isolated liver
plasma membrane vesicles (Quintana et al., 1988), it is
doubtful whether these systems allow accurate kinetic analysis of the
transporter itself (see Poole and Halestrap(1993)). Isolated
hepatocytes are more suitable and have been used in this and other
laboratories (Monson et al., 1982; Fafournoux et al.,
1985a; Edlund and Halestrap, 1988; Metcalfe et al., 1986).
However, to resolve the true initial rates of transport, we have found
it necessary to work at low temperatures (0 °C) and to measure
transport over very short time periods (Edlund and Halestrap, 1988). At
more physiological temperatures, the rapid and extensive uptake of
labeled lactate may well represent metabolism rather than transport.
The limited studies on the kinetics and substrate and inhibitor
specificity of the hepatocyte monocarboxylate transporter performed in
this way have confirmed that its properties are similar to those of
MCT1. However, using immunofluorescent microscopy, Garcia et
al.(1994) have shown that Syrian hamster hepatocytes contain
little or no MCT1. Very recently, these workers have cloned and
sequenced an MCT-related cDNA derived from hamster liver and have shown
that when expressed in insect Sf9 cells using baculovirus, it
stimulated pyruvate transport activity. This new isoform of MCT, named
MCT2, is 60% identical to MCT1, and its transport activity is inhibited
by phloretin and
-cyano-4-hydroxycinnamate (CHC) but apparently
not by organomercurials. However, only very limited functional studies
of MCT2 were performed, and it is clearly important for the properties
of the new isoform to be fully characterized.
In this paper, we have used the intracellular pH-sensitive fluorescent dye 2`,7`-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to provide such a detailed analysis of the kinetics and substrate and inhibitor specificity of monocarboxylate transport in rat hepatocytes as we have done previously in Ehrlich Lettré tumor cells (Carpenter and Halestrap, 1994) and isolated cardiac myocytes (Wang et al., 1994, 1995). Since fluorescence can be measured continuously with a very rapid time resolution, the technique produces both more accurate measurements of transport rates and allows them to be made at higher, more physiological temperatures and with any substrate. Our results confirm that the kinetics and substrate and inhibitor specificity of MCT in liver cells are similar to those of MCT1 but with some significant differences that may relate to its biological function or regulation. We also demonstrate that the transporter is capable of transporting L-lactate at rates considerably faster than required for maximum rates of metabolism, implying that the carrier is unlikely to be a major site of regulation of lactate metabolism under physiological conditions.
Figure 1:
Calibration of the BCECF 500/450
fluorescence signal in terms of protons translocated across the plasma
membrane of liver cells. Measurements of the 500/450 fluorescence ratio
of hepatocyte suspensions were determined as described under
``Experimental Procedures.'' The maximum change in
fluorescence ratio was determined following addition of the
concentrations of butyrate () or L-lactate (
)
shown. Typical traces from which such data for L-lactate were
obtained are shown in Fig. 2. Data are presented as means
± S.E. (error bars) of three experiments, each
performed on a separate hepatocyte preparation. The lines drawn are
fitted by least squares regression analysis to a hyperbolic function
with additional linear component (y = a
x/(b + x) + c
x). The initial slopes were calculated by
interpolation and used to convert rates of fluorescence ratio change to
rates of lactate uptake as described in the
text.
Figure 2: Time courses of the 500/450 fluorescence ratio changes observed with increasing concentrations of L-lactate. Measurements of the 500/450 fluorescence ratio of hepatocyte suspensions were determined as described under ``Experimental Procedures.'' In a, real-time traces of the fluorescence ratio are shown, with L-lactate being added where indicated and at the concentration shown. In the lower four traces, 5 mM CHC was added to the cell suspension 2 min before addition of L-lactate. In b, the data of a are fitted by first order regression analysis as described under ``Experimental Procedures'' section to yield initial rates of transport.
The
possibility that pH regulatory processes might lead to an underestimate
of the true rates of transport using this technique was considered.
However, the absence of bicarbonate in the buffer prevented
bicarbonate-dependent processes operating, while interference by the
Na/H
antiporter appeared minimal
since addition of 0.1 mM dimethyl-amiloride was without effect
on the initial rate of change fluorescence ratio, although the return
of pH
to normal levels following monocarboxylate-induced
acidification was slowed by its presence (data not shown).
Figure 3:
The concentration dependence of the
initial rate of fluorescence ratio change induced by L-lactate
and acetate. Initial rates of the increase of the 500/450 fluorescence
ratio induced by increasing concentrations of acetate (,
) or L-lactate (
,
) in the absence (
,
) or presence (
,
) of 5 mM CHC were
measured as described in Fig. 2. Data are presented as means
± S.E. (error bars) of three experiments, each
performed on a separate hepatocyte preparation whose loading with BCECF
was similar. In the case of L-lactate, these experiments were
the same as those shown in Fig. 1. The data for L-lactate in the absence of CHC were fitted by non-linear
least squares regression analysis to the Michaelis-Menten equation to
give values for K
and V
(±S.E.) of 5.82 ± 0.41 mM and 7.55
± 0.23 mV s
, respectively. For acetate, data
in the presence of CHC were analyzed by linear regression, which gave a
slope (±S.E.) of 0.090 ± 0.002 s
. The
data in the absence of CHC were analyzed by least squares regression
analysis to the equation v = (V
[S]/(K
+ [S])) + k
[S], where k is the slope
of the linear regression and is taken to represent the first order rate
constant for transport by diffusion of the undissociated acid as
described in the text. K
and V
values are (±S.E.) 5.45 ± 1.15
mM and 3.39 mV s
. Data from additional
experiments with different BCECF loading are included in Table 1.
Table 1presents the K and V
values for a
variety of different monocarboxylates determined in the same manner as
described for L-lactate. In each case, the initial rate of
change of the 500/450 fluorescence ratio was determined by first order
analysis for at least eight substrate concentrations, where possible
distributed at either side of the K
. The resulting
data were fitted to the Michaelis-Menten equation, and the best-fit
values of K
and V
were
derived. The data in Table 1are the means ± S.E. of these
values from the number of experiments shown, each involving a different
liver cell preparation. For comparison, data are also presented for the
kinetics of MCT1 from Ehrlich Lettré tumor cells
obtained under similar conditions (Carpenter and Halestrap, 1994). For
all but two (acetate and 4-hydroxybutyrate) of the wide variety of
substrates shown in Table 1, transport into hepatocytes was
inhibited >90% by 5 mM CHC even at the highest substrate
concentrations used, confirming that almost all transport was via the
monocarboxylate transporter. Thus, diffusion rates could be ignored.
However, for acetate and 4-hydroxybutyrate, it was found necessary to
correct for diffusion of the undissociated acid before fitting data to
the Michaelis-Menten equation. This is illustrated in Fig. 3,
where the rate of acetate uptake in the presence of 5 mM CHC
is seen to increase linearly with the acetate concentration, indicative
of free diffusion. This is to be expected, since the high
pK
of acetic acid (4.8) and the lipid solubility
of the undissociated acid support rapid rates of diffusion of acetic
acid across phospholipid membranes. In this case, data could be
successfully fitted to the Michaelis-Menten equation by subtracting the
CHC-insensitive rate as indicated in Fig. 3, and it is the
kinetic parameters derived in this manner that are reported in Table 1. In the case of propionate, the rate of diffusion
(CHC-insensitive) was so great that it was impossible to determine
accurately the kinetics of transport via the monocarboxylate
transporter.
2-Hydroxy-2-methylpropionate was found to be a poor
substrate with a low V and high K
. Other poor substrates were formate, glyoxylate,
and oxamate, and this is reflected in the large errors in their derived K
and V
values. The
branched chain monocarboxylates
-ketoisovalerate (KIV) and
-ketoisocaproate (KIC) were also tested as substrates but were
found to be transported very poorly, as shown in Fig. 4. Indeed,
an initial burst of transport was followed by a very slow rate of
transport, which was also associated with an inhibition of lactate
transport as shown by the poor response to 5 mM lactate added
subsequently (Fig. 4). Addition of increasing concentrations of
KIC and KIV prior to addition of 5 mML-lactate
allowed K
values of these monocarboxylates to be
determined as shown in Fig. 5. The mean K
values determined from three separate experiments were 270
± 28 and 342 ± 40 µM, respectively, while in
a single experiment the value for phenylpyruvate was 4.78 mM.
Figure 4: The effects of KIC and KIV on L-lactate transport into hepatocytes. Additions of 5 mM KIV, KIC, and L-lactate were made as indicated.
Figure 5:
Inhibition of the initial rate of L-lactate transport into hepatocytes by increasing
concentrations of KIC and KIV. Hepatocytes were preincubated with KIC
or KIV at the concentrations shown for 2 min before addition of 5
mML-lactate and measurement of the initial rate of
increase in the 500/450 fluorescence ratio. Data are presented as means
± S.E. (error bars) of three separate experiments. K values (±S.E.) derived by least squares
inhibition of the data shown to the equation Inhibition rate as %
control rate = 100/(1 +
[I]/K
) were 288 ± 21
and 347 ± 24 µM for KIC and KIV, respectively. Mean
values derived from the three separate experiments are given in Table 2.
Figure 6:
Inhibition of the initial rate of L-lactate transport into hepatocytes by increasing
concentrations of various inhibitors. Hepatocytes were preincubated
with the inhibitors indicated at the concentrations shown for 2 min
before addition of 5 mML-lactate and measurement of
the initial rate of increase in the 500/450 fluorescence ratio. Data
are presented as means ± S.E. (error bars) of three to
five separate experiments. K values
(±S.E.), derived from these plots as described in Fig. 5,
are given in Table 2, where K
values
derived from each separate experiments are also presented as means
± S.E.
Figure 7:
Temperature dependence of L-lactate transport into hepatocytes. In a, the
change of the 500/450 fluorescence ratio following addition of 5 mML-lactate was recorded at the temperatures indicated. In b, the initial rates of transport, calculated by first order
regression analysis of the data in a, are presented as an
Arrhenius plot that gives an activation energy (±S.E.) of 58.0
± 1.7 kJ mol.
The branched chain keto acids
KIC and KIV show an initial rapid rate of transport followed by very
slow uptake, which is associated with inhibition of the subsequent
uptake of lactate (Fig. 4) for which K values are similar to CHC (Fig. 5). This phenomenon was
also observed in tumor cells (Carpenter and Halestrap, 1994). We have
argued previously it may reflect an initial rapid carrier-mediated
entry of these monocarboxylates into the cell where some substrate,
rather than being released, forms an inactive carrier-substrate complex
involving tight binding of the substrate to a hydrophobic pocket on the
inner face of the carrier (Carpenter and Halestrap, 1994). Previous
data from other laboratories have suggested the existence of both
sodium-dependent and independent transport mechanisms for KIV and KIC
in freshly isolated and cultured hepatocytes, both inhibitable by
pyruvate (Kilberg and Gwynn, 1983; Nalecz et al., 1984).
However, the relationship between these earlier studies using
radiotracer techniques and the present ones is not obvious.
Of the
other inhibitors tested, none was found that inhibited either isoform
specifically, but there were differences observed in some of the K values. In particular, phloretin, niflumic
acid, and
-cyano-
-(1-phenylindol-3-yl)-acrylate were
4-5-fold less potent as inhibitors of L-lactate
transport into hepatocytes than tumor cells, while CHC was about half
as effective. In contrast, 3-isobutyl-1-methylxanthine was a more
potent inhibitor of transport into hepatocytes. The stilbene
disulfonates DIDS, SITS, and DBDS have similar K
values for the two transporters as does pCMBS.
Taken together,
our results suggest that the MCT present in rat liver cells is similar
but distinct from MCT1. The recent cloning of MCT2 from hamster liver
cells supports this conclusion (Garcia et al., 1995). However,
the properties of MCT2 expressed in insect Sf9 cells using the
baculovirus expression appear to show some differences to those
observed in the present paper. In particular, MCT2 appeared to be
insensitive to organomercurials unlike MCT1 and to have a higher
affinity for CHC and pyruvate than MCT1. This is clearly inconsistent
with the results presented here. Furthermore, the K values for pyruvate and the K
values for
CHC and phloretin were severalfold higher than reported here. These
differences may reflect the different membrane environment of the
transporter in the insect cells but may also be a consequence of the
radioactive transport assay used by Garcia et al.(1995). They
utilized the accumulation of 0.5 mM [
C]pyruvate after 1 min by transformed
cells; this is likely to represent the combination of pyruvate
transport and metabolism (see Poole and Halestrap(1993)). If
significant metabolism of the labeled pyruvate were to occur over this
time period, this technique would not provide a reliable measurement of
initial rates of transport. Although we cannot be certain that the
kinetics we observe with isolated rat hepatocytes represent rat MCT2,
Garcia et al.(1995) were unable to demonstrate the presence of
any MCT1 in hamster liver cells using an MCT1-specific antibody. Thus,
it seems probable that the kinetics we describe represent those of MCT2
in its natural environment.
From the temperature dependence of the transporter, the
maximum rate of transport at 37 °C can be estimated to be about 50
nmol min per µl of intracellular volume. This
translates into a transport rate of 100 nmol min
per
mg of protein, which can be compared with maximal rates of
gluconeogenesis from L-lactate in starved hepatocytes of about
8 nmol glucose min
per mg of protein (Quinlan and
Halestrap, 1986; Groen et al., 1983). Although two L-lactic acid molecules are required to make a glucose, it
would still appear that the carrier is unlikely to present a major
limitation on the rate of gluconeogenesis despite suggestions to the
contrary made by others (Metcalfe et al., 1986). This
conclusion is in agreement with measurements made of intracellular
lactate and pyruvate concentrations in hepatocytes under conditions of
rapid gluconeogenesis (Groen et al., 1983). Our data also
confirm the widely held view that lactate, pyruvate, and the ketone
bodies equilibrate across the liver cell plasma membrane, allowing
extracellular lactate/pyruvate and
-hydroxybutyrate/acetoacetate
ratios to be used to estimate cytosolic and mitochondrial
NADH/NAD
ratios, respectively (see Tischler et
al.(1977), Groen et al.(1983), and Poole and
Halestrap(1993)).