Diabetes Pharmacology Unit, Novartis Pharmaceuticals Corporation,
Summit, New Jersey 07901
A lactate clamp method has been developed to
quantify the whole body lactate utilization in conscious, unstressed
rats. Dichloroacetate (DCA), a known lactate utilization enhancer, was
used to validate the method. Fasting blood lactate concentrations
before the clamps were identical for DCA-treated (1 mmol/kg) and
control groups (1.65 ± 0.37 vs. 1.65 ± 0.19 mM). The animals
received a primed continuous lactate infusion for 90 min at variable
rates to clamp the blood lactate concentration at 2 mM. The
steady-state (60-90 min) lactate infusion rate, which represents
the whole body lactate utilization in DCA-treated animals, was 144%
higher than that in the control animals (13.2 ± 1.0 vs. 5.4 ± 1.1 mg · kg
1 · min
1;
P < 0.001). The markedly increased
lactate infusion rate indicates an enhanced lactate flux by DCA. To
determine whether the increased lactate infusion by DCA reflected
reduced endogenous lactate production, lactate production was measured.
The results indicate that endogenous lactate production was not
affected by DCA. In conclusion, the lactate clamp provides a sensitive
and reliable method to assess lactate utilization in vivo, a dynamic
measurement that may not be clearly demonstrated by blood lactate
concentrations per se.
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INTRODUCTION |
LACTATE IS BOTH an end product and a precursor of
glucose metabolism. Lactate formed from glycolysis must be converted
back to pyruvate before it can be metabolized. In a lactate-generative tissue such as skeletal muscle, lactate is either oxidized after being
converted into pyruvate or released from the cells into the blood
stream to be taken by other tissues, such as the liver. In the liver,
lactate is oxidized to pyruvate, which is then converted into glucose
via the gluconeogenic pathway. This lactate-associated gluconeogenic
process, known as the Cori cycle (5), plays an important role in
glucose homeostasis under postabsorptive conditions in healthy
individuals but also contributes to hyperglycemia in diabetes mellitus
(4, 7). Thus a knowledge of lactate utilization is important for a
better understanding of carbohydrate metabolism. Although lactate
utilization is somewhat reflected by the blood lactate concentration,
the latter is simply the balance between the lactate production and
utilization and may not adequately indicate the flux of lactate in
either direction (3). Therefore, lactate utilization cannot be
determined adequately from blood lactate concentrations per se. With
the use of an isotope tracer technique, lactate turnover rate has been
assessed in many studies (1, 10, 18). Whether the tracer technique
truly measures lactate turnover rate remains controversial (12, 13).
Furthermore, the method provides little information about the capacity
to utilize lactate. To measure such ability directly, a lactate clamp
method was developed in the present study in conscious, unstressed
animals to determine the whole body lactate utilization during a steady state of lactate infusion. Dichloroacetate (DCA) is a known enhancer of
lactate utilization. DCA activates pyruvate dehydrogenase
(PDH) by inhibiting PDH kinase, which deactivates PDH via
phosphorylation (15). The activation of PDH accelerates the
decarboxylation of pyruvate to form acetyl-CoA and thus facilitates the
conversion of lactate into pyruvate in the tissues and, therefore,
increases lactate utilization (17). In the present study, DCA was used to demonstrate the enhancement of lactate utilization and thereby validate the method. The results from this study illustrate that the
lactate clamp is a sensitive and reliable method to assess lactate
utilization in vivo.
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MATERIALS AND METHODS |
Male Sprague-Dawley rats (Hilltop, Scottdale, PA) at the age of 4 wk
were housed in hanging wire-bottom cages and given access to water and
food (diet containing 59% saturated fat, 20% carbohydrate, and 21%
protein in calories) ad libitum. All procedures described below were
approved by the Novartis Animal Care and Use Committee.
Cannulation procedure.
At the age of 10 wk, the animals were vascularly cannulated for the
study. The animals were anesthetized intraperitoneally with ketamine
(67 mg/kg; Fort Dodge Laboratories, Fort Dodge, IA) and xylazine (6.7 mg/kg; Miles, Shawnee Mission, KS). Cannulas made from Micro-Renathane
implantation tubing (0.033 in. OD × 0.014 in. ID and 0.040 in. OD × 0.025 in. ID; Braintree Scientific, Braintree, MA)
were implanted into right jugular vein and left carotid artery and
filled with saline containing 35% polyvinylpyrrolidone and 1,000 U/ml
heparin. The cannulas were exteriorized at the nape of the neck and
anchored to the skin.
Lactate clamp procedure.
Five days after the surgery, the animals were randomly assigned to
control or DCA treatment groups (n = 4/group). The animals were fasted at 10:30 AM on the day before the
lactate clamp study. At 7:45 AM on the day of the study, the animals
were given dosing vehicle (0.5% carboxymethylcellulose with 0.2%
Tween 80; Sigma, St. Louis, MO) or DCA (1 mmol/kg; Aldrich, Milwaukee,
WI) in vehicle by oral gavage (10 ml/kg). At 9:30 AM, the animals were
placed in plastic metabolic cages after their cannulas were connected to blood sampling tubing or lactate infusion syringes mounted on
infusion pumps (Harvard Apparatus Syringe Infusion Pump 22, Harvard
Apparatus, South Natick, MA). The lactate infusate contained 20%
sodium lactate in saline, and the pH was adjusted to 7.0 with 20%
lactic acid in saline (Sigma, St. Louis, MO). At 10:20 AM, a blood
sample (20 µl) was taken via the arterial cannula for the measurement
of fasting blood lactate and glucose concentrations before the clamp (0 h). After the 0-h blood sample was taken, 0.2 ml of heparinized saline
(100 U/ml) was injected via sampling line to prevent blood clotting in
the line during the clamp. At 10:30 AM, a primed lactate infusion at 56 mg · kg
1 · min
1
was given for 2 min via the venous cannula to quickly raise blood lactate levels. After the primed infusion, a constant lactate infusion
at 2 mg/min was given for 3 min before an adjustment of infusion rate
was made based on blood lactate levels. Blood samples were taken via
the arterial cannula at 5-min intervals for lactate measurement and at
15-min intervals for glucose measurement during the 90-min clamp. Blood
lactate levels were maintained at ~2 mM via the adjustment of lactate
infusion rates. Blood lactate concentrations were determined
immediately after sample collection using an Analox GM7 Micro-Stat
multi-assay analyzer (Analox Instruments, London, UK), and blood
glucose concentrations were determined using an Analox GM9 glucose
analyzer. The blood sample amounts are required for the lactate and
glucose assays are 7 and 10 µl, respectively, and the total blood
loss due to sampling during the clamp was <0.3 ml.
Measurement of lactate production.
In separate experiments, the effect of DCA treatment on endogenous
lactate production was determined in the animals that received identical diet, cannulation surgery, fasting, and dosing treatment to
those in lactate clamp experiments. The animals
(n = 5 for control group,
n = 6 for DCA group) were infused with
L-[14C(U)]lactate
(Du Pont, Wilmington, DE). A bolus infusion of 5 µCi of
[14C]lactate was
administered within 2 min, followed by a constant infusion at 0.1 µCi/min for 43 min. Blood samples (150 µl each) were collected at
30, 40, and 45 min for determination of plasma lactate concentrations
and 14C counts. The blood samples
were immediately centrifuged in a refrigerated centrifuge at 1,000 g for 10 min to separate the plasma.
Fifty microliters of plasma were mixed with 100 µl of 10% TCA and
then centrifuged to obtain the supernatant. Fifty microliters of
supernatant were aliquoted into a scintillation vial, in duplicate, to
which 5 ml of scintillation fluid were added and then counted for 10 min in a Beckman scintillation counter (model LS 3801; Beckman
Instruments, Irvine, CA).
Data analysis.
Data are reported as means ± SE. The average lactate infusion rate
(mg · kg
1 · min
1)
at the steady state (60-90 min) of the clamp was calculated and
used as an index for the whole body lactate utilization. The average
specific activity of plasma
[14C]lactate (dpm/mg)
during the last 15 min of
[14C]lactate infusion
experiments was calculated and used to determine the rate of lactate
production
(mg · kg
1 · min
1),
which was calculated as the rate of tracer lactate infusion (dpm/min)
divided by the plasma specific activity of lactate (1).
Statistical analysis was performed to compare the control and
DCA-treated groups with the use of a two-way ANOVA with repeated measures for blood lactate and glucose levels and lactate infusion rates during the clamps, and an unpaired
t-test was performed for fasting blood
lactate and glucose levels before the clamps, the average lactate
infusion rates at the steady state, and the lactate production rates.
Statistical significance was accepted at
P < 0.05.
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RESULTS |
Body weights and fasting blood lactate and glucose concentrations
before the clamps are shown in Table 1.
Fasting blood lactate and glucose levels measured before the clamps
were almost identical for both groups. Blood lactate concentrations for
the control and DCA-treated groups during the clamps are shown in Fig.
1. Although the blood lactate
concentrations before the clamps were not different between the two
groups, the same amount of lactate infusion for the first 5 min of the
clamp caused a more rapid increase in blood lactate level in the
control group compared with the DCA-treated group. Blood lactate levels
during the clamps for both groups were maintained around 2 mM,
indicating that the target lactate level of the clamp was achieved.

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Fig. 1.
Blood lactate levels before and during lactate clamps in control and
dichloroacetate (DCA)-treated rats. Values are means ± SE for 4 animals/group.
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Lactate infusion rate during the clamps was markedly higher in
DCA-treated animals compared with the control rats
(P < 0.01) (Fig.
2). The significant difference in lactate
infusion rates between the two groups was maintained throughout the
clamps. The average lactate infusion rate from 60 to 90 min is shown in
Fig. 3. The average lactate infusion rate
during that period of the clamp reflects the whole body lactate
utilization rate at a steady state. In DCA-treated rats, the average
lactate infusion rate was 144% higher than that in the control rats
(P < 0.001).

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Fig. 2.
Lactate infusion rates during lactate clamps in control and DCA-treated
rats. Values are means ± SE for 4 animals/group. Infusion rates
during clamps were significantly different between 2 groups
(P < 0.01).
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Fig. 3.
Average lactate infusion rate from 60 to 90 min during lactate clamps
in control and DCA-treated rats. Values are means ± SE for 4 animals/group. * P < 0.001 vs.
control group.
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Figure 4 shows blood glucose concentrations
during the clamps. Although blood glucose concentrations before the
clamps were not different between the control and DCA-treated groups,
15 min after the onset of the clamp blood glucose level in the control animals was elevated and maintained at a higher level during the rest
of the clamps. In contrast, blood glucose level in DCA-treated animals
was maintained at the basal level until the end of the clamps. There
was a significant difference between the blood glucose levels of the
control and DCA-treated groups during the clamps (P < 0.05). The ANOVA,
however, showed that the drift of the glucose levels during the clamps
was not statistically significant.

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Fig. 4.
Blood glucose concentrations before and during lactate clamps in
control and DCA-treated rats. Values are means ± SE for 4 animals/group. Glucose concentrations during clamps were significantly
different between 2 groups (P < 0.05).
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In the study for the measurement of lactate production, blood lactate
levels at 0 and 45 min were, respectively, 1.46 ± 0.04 and 1.44 ± 0.09 mM for the control group and 1.35 ± 0.07 and 1.38 ± 0.07 mM for the DCA-treated group. The difference between the two
groups in blood lactate levels at both time points was not statistically significant. Lactate production rates determined during
the last 15 min of
[14C]lactate infusion
experiments are shown in Fig. 5. There was no significant difference in lactate production between the DCA-treated and the control groups.

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Fig. 5.
Lactate production rate in control and DCA-treated rats. Values are
means ± SE for 5 or 6 animals/group.
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DISCUSSION |
Blood lactate concentration measurement has been frequently used as an
indicator of lactate metabolism in vivo. Although the measurement is
simple and provides useful information, it does not adequately reflect
either lactate production or utilization, because blood lactate level
is only a set point at which lactate production and utilization reach a
balance. The present study has established the lactate clamp method to
measure the whole body lactate utilization. The results from this study
show that lactate utilization can be measured adequately by the clamp
method, as demonstrated by a significant enhancement of lactate
infusion rate in animals treated with DCA compared with the animals in the control group. This significantly higher lactate infusion rate was
maintained throughout the clamp, providing a clear indication of
enhanced lactate utilization.
To clamp the blood lactate at a desired level, it is necessary to
rapidly raise and then maintain the blood lactate at a level that is
higher than the basal lactate concentration. A very mild hyperlactatemia was achieved in the present study, which is within the
physiological range of blood lactate. The 21% increase of blood
lactate level from 1.65 mM at basal to 2.0 mM during the clamp is much
smaller than the lactate elevations seen in studies using constant
lactate infusion methods, in which lactate concentrations were raised
to at least two- to fourfold of the basal concentration (8, 19). The
minimal hyperlactatemia applied in the lactate clamp method provides a
significant advantage over the more severe hyperlactatemia shown in the
traditional constant lactate infusion methods. First, a severe
hyperlactatemia could exert a profound effect on carbohydrate
metabolism and electrolytic/acid-base balance, and, therefore, may
affect the hormonal responses to the changes in metabolism. Second,
without a stable blood lactate level being achieved, a constant lactate
infusion may result in different levels of hyperlactatemia among
individuals, which in turn may induce different levels of lactate
removal and inhibition of endogenous lactate production and thus make
the comparisons among the individuals less meaningful. To choose an
optimal blood lactate level for the clamp, lactate clamp at 3 mM of
blood lactate was also evaluated in our pilot studies. The results
showed that the clamp at 2 mM was more sensitive to the DCA treatment
than the clamp at 3 mM (unpublished data). It is important to maintain
the experimental condition as close to the physiological condition as
possible. However, to measure lactate utilization under various
conditions, such as diabetes for example, it may be necessary to clamp
blood lactate at levels other than 2 mM.
Isotope tracer technique has been applied to the measurement of lactate
turnover rate (1, 10, 18). Lactate turnover rate is calculated by
dividing the lactate tracer infusion rate by the lactate specific
radioactivity in the blood. When a steady state is achieved during
tracer infusion, rate of lactate turnover equals rate of lactate
production and rate of lactate utilization (2). Although the lactate
tracer dilution technique has been used extensively to assess lactate
metabolism, the measurement of lactate turnover rate might be
complicated because that lactate tracer is reversibly converted into
pyruvate before being irreversibly removed from the system (13). The
total loss of labeled lactate to pyruvate in blood amounts to ~10%
of lactate disappearance at rest (12). Also, radioactivity of plasma
14CO2
derived from lactate oxidation, although in a negligible amount, may
need to be taken into consideration when the lactate turnover rate is
calculated. Various compartmental models have been proposed to modify
the calculation for the lactate turnover rates (12, 16). Although rapid
isotopic exchange between lactate and pyruvate is taken into account in
these models, the interpretation of lactate kinetic data obtained from
the use of lactate tracer should be made with caution. Unlike the
lactate tracer technique, the lactate clamp is a simple method to
measure lactate utilization during a very mild hyperlactatemia. Aside
from the simplicity of the measurement, the lactate clamp also provides
the assessment for the capacity of lactate utilization, which cannot be
determined by the tracer technique.
During the clamp, the lactate appearing in the blood may originate from
both endogenous and exogenous sources, i.e., the lactate produced from
tissues and the lactate infused. Thus the increased lactate infusion
rate during the clamps in DCA-treated animals may reflect reduced
endogenous lactate production, i.e., the lactate utilization in animals
treated with DCA might be overestimated by using lactate infusion rate
as an indicator. To investigate this possibility, the effect of DCA
treatment on endogenous lactate production was measured. The results
for lactate production rate determined from the
[14C]lactate infusion
experiments clearly show that the treatment with DCA did not alter
lactate production rate in these animals. The lactate production rate
during the clamps was not measured in the present study to keep the
clamp method relatively simple. It has been reported that infusion of
lactate at a rate of 140 mg · kg
1 · h
1
completely inhibited endogenous lactate production in humans (14). The
lactate infusion rates in the present study were much higher (averaged
at 306 ± 60 and 738 ± 36 mg · kg
1 · h
1
for the control and DCA-treated groups, respectively), and a complete
inhibition of endogenous lactate production could be expected. Although
it is difficult to compare studies using different species, in light of
the small amount of lactate production under basal condition and the
same blood lactate levels during the clamps in both groups in the
present study, even if the lactate production was not completely shut
down, it is very unlikely that the difference between the control and
DCA-treated groups in lactate production rate, if any, would have much
impact on lactate infusion rate during the clamps. Therefore, the
lactate infusion rate during the clamps may closely represent the rate
of the whole body lactate utilization. There is a controversy about the
lactate turnover measurement using arterial (venous-arterial procedure)
or venous (arterial-venous procedure) lactate specific
activity (1, 10). We did not compare the two procedures in the present
study. However, the purpose of the lactate turnover measurement was to
address whether there was a difference between the control and
DCA-treated groups. The outcome may not be affected by using either
procedure, although values obtained from the two procedures may be
different.
Although DCA is known to increase lactate utilization by its inhibition
of PDH kinase, as demonstrated by increased PDH activity in various
tissues (17), the effect of DCA on blood lactate concentration in
animals was not always apparent. We have routinely observed that the
decrease in blood lactate concentration in DCA-treated (1 mmol/kg,
maximally effective dose) rats is no more than 30%, and the effect of
DCA usually peaks between 2 and 4 h after the treatment (unpublished
data). In the present study, the blood lactate concentration before the
clamp was not even affected by the DCA treatment. Thus measuring blood
lactate concentration per se is not a reliable way to assess lactate
utilization. Despite the lack of effect on blood lactate concentration
before the clamps, the DCA treatment markedly increased lactate
infusion rate by 2.4-fold during the clamps. The significant and
consistent stimulation of lactate removal during the clamps after the
DCA treatment suggests that lactate flux is greatly enhanced by the
treatment. It also suggests that lactate infusion rate during the
clamps is a sensitive and reliable measurement for assessing lactate
utilization in vivo.
In addition to the change in lactate infusion rate, blood glucose
concentration in DCA-treated animals during the clamps was significantly lower than that in the control animals, despite the
similar blood glucose levels for the two groups before the clamps. It
has been hypothesized that lactate formed in one tissue could
distribute through interstitium and circulation to other tissues to be
oxidized or used for other metabolic processes, such as
gluconeogenesis, a lactate shuttle theory supported by several lines of
evidence (3). A lower blood glucose level and a greater lactate
infusion rate during the clamps in the DCA-treated group suggest that
the excess lactate was oxidized rather than being used as a precursor
for gluconeogenesis in the liver. It has been reported that DCA
effectively inhibits gluconeogenesis from lactate in isolated
hepatocytes by limiting substrate availability as well as the direct
inhibition from oxalate, a metabolite of DCA (6). Although hepatic
glucose production during the lactate clamps was not measured, our
recent study measuring hepatic glucose production after an oral lactate
administration showed that DCA treatment prevented a significant
elevation of hyperlactatemia-stimulated hepatic glucose production in
rats (9). Thus, when combined with the measurement of hepatic glucose
production during the clamps, the lactate clamp method may provide a
useful assessment for the status of the Cori cycle, especially during
starvation, or with certain metabolic disorders, such as diabetes
mellitus (4, 7). The lactate clamp may also be useful to elucidate the
mechanism of the hyperlactatemia associated with liver damage, such as
reduced hepatic perfusion, hepatocyte failure, hepatic tumor, genetic
gluconeogenic enzyme deficiency, or other dysfunctions (11).
In conclusion, the lactate clamp provides a sensitive and reliable
method to assess in vivo lactate utilization, a dynamic measurement
that may not be clearly demonstrated by blood lactate concentrations
per se.
Address for reprint requests: J. Gao, LSB 3505, Novartis
Pharmaceuticals Corp., 556 Morris Ave., Summit, NJ 07901.