2 Département de Physiologie, Laboratoire de Physiologie des Interactions, Institut de Biologie, and 1 Département de Biochimie Métabolique et Clinique, Faculté de Pharmacie, 34060 Montpellier, France
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ABSTRACT |
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The present experiments were undertaken to characterize 1) the hindlimb muscle mass lactate uptake and 2) the expression of monocarboxylate transporter isoforms MCT1 and MCT4, as well as lactate dehydrogenase (LDH) isozyme distribution, in various skeletal muscles of Zucker fa/fa rats taken as a model of insulin resistance-related obesity. Initial lactate uptake at six different concentrations was measured in sarcolemmal vesicles (SV) by use of L-[U-14C]lactate. Compared with controls, the maximal rate of lactate uptake and affinity were decreased in SV of Zucker rats (~30%) in which MCT4 content was significantly decreased (P < 0.05). MCT4 expression was decreased in soleus, extensor digitorum longus, and red tibialis anterior (RTA; P < 0.05), but not in white tibialis anterior, whereas MCT1 expression was decreased only in RTA of Zucker rats (P < 0.05). Obesity led to a shift toward type M-LDH isozyme in mixed muscles. We conclude that obesity leads to changes in muscular MCT1 and MCT4 expression, which, when associated with LDH isozyme redistribution, may contribute to the hyperlactatemia noted in insulin resistance.
monocarboxylate transporter; muscle fiber type; lactate transport; lactate dehydrogenase isozyme; Zucker fa/fa rat
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INTRODUCTION |
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LACTATE PRODUCTION is a metabolic event that is receiving increasing attention, because peripheral conversion of glucose into lactate plays a significant role in the synthesis of liver glycogen via the gluconeogenic pathway after glucose ingestion (27). Lately, it has become evident that adipose tissue is an important source of lactate production in vivo (19) and that this process is enhanced by both insulin and catecholamine stimulation (20). Interestingly, a significant inverse correlation between overnight fasting lactate level and insulin sensitivity has been found in obese subjects (26), and this correlation is stronger than the relationship between lactate levels and body mass index in this population. Moreover, it has been observed that patients with type 2 diabetes have even greater elevations in basal lactate levels than do obese subjects (34). This association between elevated basal lactate levels, insulin resistance, and diabetes is supported by epidemiological studies in healthy subjects in whom an elevated fasting lactate level was found to be a significant and independent risk factor for the development of type 2 diabetes (32).
Quantitatively, skeletal muscle mass appears to be by far the most important insulin-sensitive tissue, indeed, far more important than liver and adipose tissue. On the other hand, this insulin-sensitive skeletal muscle mass can both produce and utilize lactate as a fuel for mitochondrial oxidation relative to fiber type, and it presents a net lactate release in the resting condition in normal subjects (8). In this respect, it is tempting to implicate skeletal muscle mass in the mechanisms leading to basal hyperlactatemia through an increased lactate production/interconversion rate with pyruvate, as previously described (1), impaired fiber type-dependent lactate uptake and oxidation, or both mechanisms.
In the past few years, it has been acknowledged that skeletal muscles and most other tissues have a membrane transport system mediating a coupled lactate and proton translocation (33). Moreover, in muscle, several lactate-proton monocarboxylate transporter isoforms (MCTs) are coexpressed, two of which have already been cloned: MCT1 and MCT4 (33). Whether an impaired lactate exchange across key plasma membrane organs is involved in obesity and the insulin-resistant state and whether this contributes to reduced lactate clearance have not yet been evaluated. Moreover, a potential redistribution in lactate dehydrogenase (LDH) isozymes that could participate in the previously described increased lactate/pyruvate interconversion rate has also not been investigated. Therefore, this study aimed to 1) characterize the hindlimb muscle mass lactate uptake by use of the model of sarcolemmal vesicles coupled with the expression of MCT1 and MCT4 and 2) determine LDH distribution in various skeletal muscles of insulin-resistant obese Zucker fa/fa rats.
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MATERIALS AND METHODS |
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Reagents. Reagents were purchased with the highest quality available from Sigma Chemical (L'isle d'Abeau Chesnes, St Quentin Fallavier, France) unless otherwise stated.
Animals. Animal experimentation was performed according to the Helsinki convention for animal care and use. Male Zucker fa/fa and Wistar rats, 11 wk old, were purchased from IFFA CREDO (Charles River, l'Abresle, France). They were kept on a reverse 12:12-h light-dark cycle (lights on at 7 PM) at 22°C and housed in individual cages. Standard rat chow and water were provided ad libitum.
Two weeks after their arrival in the laboratory (13 wk old), blood samples for determination of plasma glucose, lactate, and insulin were collected in the nonfasting state from rats of each group before euthanasia. One Zucker rat and one control rat were then killed on the same day by cervical dislocation for tissue preparation.Tissue preparation.
After cervical dislocation, hindlimb muscles were rapidly removed.
Portions of red and white tibialis anterior (RTA and WTA, respectively), extensor digitorum longus (EDL), and soleus (SOL) were
quickly frozen in liquid nitrogen and stored at 80°C until biochemical assays were performed. The remaining hindlimb muscles were
used for sarcolemmal isolation.
Sarcolemmal vesicle isolation and characterization.
Sarcolemmal vesicles (SV) were purified from hindlimb muscles with a
procedure already described (10, 11, 14). Proteins were
determined according to the procedure of Bradford with bovine -globulin as a standard. SV characterization was achieved with K+-stimulated p-nitrophenylphosphatase
(K+-pNPPase) assay, as previously described
(10, 11). The purification index (PI) was defined as the
ratio of the specific activity from the sarcolemmal fraction (SF) to
the specific activity measured in the crude homogenate (CH). Skeletal
muscle SV yield was the ratio of sarcolemmal proteins (mg) obtained in
SF to the muscle weight in grams after trimming (wet weight).
Lactate transport studies. All measurements were performed in zero-trans conditions and in duplicate. L-[U-14C]lactate (sodium salt) was purchased from Amersham (specific activity 155 mCi/mmol) and was diluted in 280 mM sucrose and 50 mM HEPES, pH 7.4, and different unlabeled L(+)-lactate concentrations. Reciprocal decreases in sucrose were used to maintain the same total isosmotic buffer strength (10, 11). Reactions were initiated by delivering 50 µg of protein in tracer-containing medium and then stopped at appropriate time intervals by vacuum filtration on nitrocellulose filters (Whatman WCN, average pore size of 0.45 µm). Filters were then rinsed three times with an ice-cold isosmotic medium consisting of Krebs-Henseleit (KRH) buffer with the addition of 3 mM HgCl2, pH 7.4, dissolved with ethyleneglycolmonomethylether, and the radioactivity was counted on a scintillation analyzer (Packard 2200 CA). Nonspecific transport activities were determined by preincubation of vesicles in tracer-containing medium with KRH buffer containing 3 mM HgCl2, which was used to fix the zero time points. Results were expressed in nanomoles per milligram protein. Rates of initial lactate uptake were measured for 1, 5, 10, 30, 50, 70, and 100 mM external lactate concentrations at 0 and 10 s, when uptake rates are assumed to be constant for this time period (35), and during which zero-trans conditions are respected. Slopes gave initial rates of lactate uptake (lactate uptake10s) expressed in nanomoles of lactate per minute per milligram protein. We also measured the time course of zero-trans lactate uptake with an external lactate concentration of 1 mM. For the determination of the kinetic parameters, nonlinear regression algorithms of SIGMA-PLOT software (Jandel Scientific, Erkrath, Germany) were used. The slopes of initial lactate uptake were fitted to the appropriate equation. The Michaelis-Menten constant for substrate affinity (Km) values were determined by nonlinear regression of values to the equation v/Vmax = [S]/(Km + [S]), where v is the measured initial rate of lactate uptake, Vmax is the maximal rate of lactate uptake, and [S] is substrate concentration.
Vesicle sensitivity to osmotic forces. The effect of medium osmolarity on L-(+)-lactate uptake at equilibrium in SV was determined by using different external media containing 1 mM L-(+)-lactate with the tracer and various sucrose concentrations (150, 200, 250, 300, 350 mM in 50 mM Tris, pH 7.4). Intravesicular medium consisted of 250 mM sucrose and 50 mM Tris, pH 7.4. Equilibrium L-(+)-lactate uptake was determined after 3-min incubations, and results were expressed in nanomoles per milligram protein per 3 min.
Sample preparation for Western blotting. Proteins were isolated from muscles for Western blotting, as previously described by McCullagh et al. (29) and previously used in this laboratory (13, 14). Muscle protein concentrations were determined in triplicate by the bicinchoninic acid assay (Pierce, Interchim, Montluçon, France) with bovine serum albumin as a standard.
Western blotting of MCT1 and MCT4. Affinity-purified polyclonal antibodies directed against the carboxy terminus of rat MCT1 were produced by immunization of New Zealand White rabbits with the synthetic peptide PLQNSSGDPAEEESPV for MCT1 and LREVEHFLKAEPEKNG for MCT4 (9). Polyclonal antibodies yielded a single band on a Western blot that corresponded to 43 kDa, consistent with the molecular mass reported earlier (23, 29, 37). Antibody specificities were confirmed in preliminary experiments in which the peptides blocked the detection of MCT1 and MCT4. Protein (20 µg) of muscle homogenates and prestained molecular mass markers (Bio-Rad, Ivry-sur-Seine, France) were separated on 12% SDS-polyacrylamide gels (140 V for ~90 min). Proteins were then transferred from the gels to polyvinylidene difluoride membranes (100 V, 90 min). Membranes were incubated on a shaker overnight at room temperature in buffer D [20 mM Tris base, 137 mM NaCl, 0.1 M HCl, adjusted to pH 7.5, 0.1% (vol/vol) Tween 20, and 5% (wt/vol) nonfat dried milk]. Membranes were then incubated with diluted carboxy terminus of either MCT1 antibody (1:3,000) or MCT4 antibody (1:3,000) in buffer D for 1 h 30 min, followed by three washes in buffer E (i.e., buffer D without dried milk: 3 × 5-min washes), followed by incubation for 45 min with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (1:3,000; BI 2407, BioSys, Compiègne, France) in buffer E. Membranes were washed as previously described, and MCT1 or MCT4 expression was detected by enhanced chemiluminescence (Biomax MR films, Kodak). Films were developed and fixed using a hyperprocessor, RNP 1700 (Amersham, Les Ulis, France). MCT1 and MCT4 protein band densities were determined by scanning the blots on a scanner (AGFA Duo Scan T1200) and with Scion Image software. For MCT1, the signal of control heart preparation was used as a positive control and to fix an arbitrary unit to allow comparison between experiments (100% equals the MCT1 signal of 20 µg of control heart homogenate).
When possible, SV preparations were blotted against MCT1 and MCT4 after lactate transport experiments. Fifteen micrograms of protein were separated onto SDS-polyacrylamide gels, transferred, and immunoblotted as described above.LDH isozyme distribution. LDH isozymes present in muscle homogenates were separated by adding 1 µg of protein to agarose (1%) gels and electrophoresing at 90 V for 30 min on a Bio-Rad Sub-Cell system. An electrophoretic marker (LDH Isotrol, Sigma) containing LDH isozymes 1-5 was used as an aid in identification of isozymes. LDH isozyme activities were visualized by nitro blue tetrazolium reduction to formazan (Sigma Procedure 105). The gels were fixed in 5% acetic acid. The different bands were scanned and quantified as described above. LDH isozyme (-1 to -5) repartition was calculated by multiplying the area with the mean optic density for each isozyme and then dividing it by the sum of the product area × mean optic density of the five isozymes (the sum of each area × mean optic density product for each isozyme equal to 100%). Results are expressed as a percentage of all LDH isozymes.
Additional assays.
Plasma lactate concentration was determined using the method of Gutmann
and Wahlefeld (18). Insulin concentration was measured by
the method of Herbert et al. (21). Proteins were estimated by the Coomassie brilliant blue method using bovine -globulin as a
standard (Bio-Rad protein assay). Results are expressed in micromoles
per minute per gram protein. Blood glucose levels were determined by
the glucose oxidase method.
Data analysis. Results are expressed as means ± SE. The data were analyzed using regression analyses, t-tests, and analyses of variance when appropriate. Normality distribution of the data was checked using a Kolmogorov-Smirnov test. The differences were considered significant at P < 0.05. Pearson product-moment correlations were used to detect relationship between variables.
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RESULTS |
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Basal blood glucose, lactate, and insulin concentrations.
Basal blood glucose, lactate, and insulin concentration values are
presented in Table 1. Obese Zucker rats
were characterized by increased basal plasma insulin and lactate
concentrations, whereas plasma glucose remained unchanged. Plasma
lactate concentration increased by 48%, i.e., up to 2.22 mmol/l
(P < 0.05).
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Characterization of SV.
Using gradient density fractionation, we obtained SV preparations with
similar biochemical characteristics in both groups. A slight but
significant increase in the SF enrichment in the Zucker group
was observed (0.16 ± 0.02 vs. 0.11 ± 0.01 mg/g protein, P < 0.05). However, there was no significant
difference in the purification indexes of
K+-pNPPase between the control and
Zucker groups (Table 2).
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Lactate transport kinetics.
Figure 1 shows the lactate
uptake10s in vesicles from the control and Zucker
fa/fa groups plotted as a function of external lactate
concentration. SV from obese rats showed a general decrease in total
lactate influx rates compared with control rats. Initial rates
displayed saturation kinetics with Vmax values
of 340 and 259 nmol · min1 · mg
protein
1 and Km values of 30 and
41 mM for control and Zucker groups, respectively. Thus the maximal
rate of lactate influx into SV from the Zucker rats was decreased by
nearly 34%, whereas the affinity for the substrate decreased by 37%.
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Effect of osmotic forces.
Figure 2 shows the effect of osmolarity
on 1 mM L-(+)-lactate uptake at equilibrium in SV from the
control and Zucker groups. A two-way ANOVA revealed that decreasing the
intravesicular space by higher external sucrose concentration resulted
in less L-(+)-lactate accumulation in vesicles from the two
groups at pH 7.4 (P < 0.05). Moreover, lactate uptake
at equilibrium was significantly higher in the control than in the
Zucker group (P < 0.05). Thus, whereas osmotic
pressure exerted the same effect on the two groups, a more pronounced
decrease in lactate transport in SV from Zucker rats was observed when
compared with SV from control rats.
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MCT1 and MCT4 expression in skeletal muscles.
In control and Zucker rats, MCT1 was detected in the four skeletal
muscles studied (Fig. 3A).
Densitometry revealed the same MCT1 concentration profile in both
groups: SOL contained the highest level of MCT1, followed by RTA, EDL,
and WTA muscles (Fig. 3B). Relative to control heart as the
positive standard, there was a trend toward a reduced MCT1 expression
in SOL, EDL, and WTA muscles of the Zucker group (45.4, 11.6, and 3.9%
for SOL, EDL, and WTA, respectively, in the control group and 35.5, 10.7, and 2.3%, respectively, in the Zucker group). However, a
significant decrease was observed only in RTA of the Zucker rats (17 vs. 32.1%, P < 0.05; Fig. 3B).
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MCT1 and MCT4 expression in SV.
When possible, we performed immunoblots of SV preparations from both
groups. Figure 5 shows that the number of
MCT4 transporters present in the SV of Zucker rats was decreased
compared with controls, whereas only a trend toward the decreased MCT1
transporter was noted.
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LDH isozyme distribution.
The LDH isozyme distribution in skeletal muscles is represented in
Table 3. All LDH isozymes (LDH-1 to
LDH-5) were detected in SOL, EDL, and RTA muscles, whereas only LDH-3,
-4, and -5 were detected in WTA of the control and Zucker rats. The
profile of the relative percentage of each isoform in SOL, with LDH-1
and LDH-2 predominant, differed from that of the EDL, RTA, and WTA muscles, in which LDH-5 was present in the highest amount. Obesity led
to changes in the relative percentage of isozyme distribution in the
Zucker rats, with differences between muscles relative to their
oxidative and glycolytic capacities. A 14% decrease was noted in the
LDH-2 isozyme in SOL, although this was not significant. No change was
found in the more glycolytic WTA muscle. For EDL and RTA muscles we
observed similar changes, with the LDH-1 decrease more pronounced in
RTA (70%, P < 0.05) than in EDL (53%,
P < 0.05). LDH-5 was also decreased by 18 and 15%
(P < 0.05) in RTA and EDL, respectively. Moreover,
there was a 61% increase in the intermediate isozymes LDH-3 and LDH-4
for both muscles and a 35 and 51% increase for LDH-4 in RTA and EDL,
respectively.
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Relationship between MCT1 and LDH isozyme distribution.
Using Pearson product-moment correlations, a positive relationship in
the two groups appeared between their MCT1 and LDH-3 isozyme content
(r = 0.95 and 0.98 for control and Zucker groups, respectively, P < 0.05; Fig.
6) and between MCT1 and the LDH-2 isozyme
(r = 0.96, P < 0.05) in the Zucker
group. Surprisingly, we were not able to find the same positive
correlations between MCT1 content and heart-type isozymes (LDH-1 and
-2) as previously described in the control rats (29).
Nevertheless, a strong correlation appeared between LDH-1 and -2 in
both groups (r = 0.99 and 0.98 for the control and
Zucker groups, respectively, P < 0.05; Fig. 6).
Moreover, comparison between MCT1 and the LDH-5 isozyme resulted in a
strong negative relationship only in the Zucker group
(r = 0.99, P < 0.05; Fig. 6).
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DISCUSSION |
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We attempted to characterize hindlimb muscle mass lactate uptake and MCT1-MCT4 expression, as well as LDH isozyme distribution, in various skeletal muscles of Zucker rats to specify the possible contribution of skeletal muscles in the basal hyperlactatemia present in this model of insulin resistance. The main results we observed in Zucker rats compared with controls are 1) a decrease in the maximal rate of lactate uptake in SV, 2) a fiber type-dependent decrease in MCT1 and MCT4 expression, and 3) an effect of obesity on the redistribution of LDH isozymes with a shift toward more type M-LDH.
Blood lactate concentration increases in many physiological and pathological conditions, such as physical exercise, fasting, type 2 diabetes, obesity, and hypertension (6). When released in the circulation, lactate may be taken up by different organs, but skeletal muscle, heart, and liver appear to be major sites of lactate removal. In resting conditions, muscle lactate utilization appears to be largely dependent on oxidation (17). Insulin resistance-related obesity in Zucker rats, like that in humans, seems to be characterized by increased resting blood lactate concentration that may be the result of both impaired lactate metabolism and impaired exchanges in skeletal muscle.
We investigated lactate transport into SV from control and Zucker rats
and found a decreased total influx rate at all external lactate
concentrations in the Zucker group. The decreased maximal rates of
lactate uptake (34%) found in the SV of these rats may represent a
decreased number of transporters present in the SF. The properties of
lactate transport in our SV were similar to the standard results
in isolated membranes (24, 31, 35). We previously reported
that our system exhibits detectable lactate transport properties,
saturation with increasing external lactate concentration, sensitivity
to pH and -cyano-4-hydroxycinnamate (a monocarboxylate transport
inhibitor), and some trans-stimulatory properties at high
external L-(+)-lactate concentration (10, 14).
Despite low sarcolemmal recovery from skeletal muscle, isolated SV
constitute a nonmetabolic system that allows detailed studies of
membrane properties. In the present study, the SV of the two groups
appeared to be sealed, because they showed identical sensitivity to
changes in osmotic conditions (Fig. 2). In equilibrium conditions, SV
from the Zucker group also showed reduced lactate accumulation. This
result could indicate that SV from the Zucker group were smaller than
those of the control group. It has been reported that when measurements
are made in initial rate conditions, the uptake is dependent only on
the transporter activity. This is unlike equilibrium conditions, where
the amount of accumulated substrate in SV is dependent on both
transporter activity and vesicle volume (22). These
findings suggest that obesity and/or insulin resistance could either
influence the sarcolemmal isolation procedure or induce membrane
alterations that might perturb lactate transport properties, resulting
in decreased lactate accumulation. Because the protein yields, although
slightly increased in the Zucker group, and the purification indexes
were quite similar in both groups, it is unlikely that obesity and/or
insulin resistance could have affected the vesicle volume and
purification of SV.
Recently, it was established that skeletal muscles and most other tissues possess a membrane transport system mediating a coupled lactate and proton translocation (33). MCT1 was the first rat and human skeletal muscle isoform to be cloned, by Garcia et al. (15). More recently, Wilson et al. (37) cloned MCT4 (formerly MCT3), which showed a strong expression in rat and human skeletal muscle. MCT4 content appears to be similar in the various skeletal muscles except for a markedly lower content in SOL, suggesting that only the fast oxidative glycolytic (FOG) fibers in SOL possess this isoform (37). In the present study, the Zucker rats had a more pronounced decrease in the expression of MCT4 than of the MCT1 isoform, relative to the fiber type composition of muscle. MCT1 expression was decreased in RTA muscle, which is composed primarily of type IIA (FOG) fibers, although only a trend toward decreased expression was noted in both slow-twitch and fast-twitch muscles; MCT4 expression was decreased in all muscles except the fast-twitch WTA muscle. These results may explain why the MCT4, but not the MCT1, content in SV prepared from the Zucker rat hindlimb muscles appeared to be decreased (Fig. 5). Furthermore, when SV are prepared, the rat hindlimb muscles that are pooled consist largely of type II fibers that contain predominantly MCT4 (5). Thus MCT4 was expected to be quantitatively the more represented isoform in the SV of both groups. Therefore, with both decreased MCT1 expression in FOG fibers and essentially decreased MCT4 expression in a wider range of muscle fiber types, the SV of Zucker rats were expected to have a decreased content in MCTs appreciated by the decreased Vmax in this group. Moreover, another clue that might provide information about the percentage of each transporter present in SV appears to be the transport affinity determined in SV. The determined Km of both groups (30-40 mM) was closely related to the recently determined Km of MCT4 when expressed in Xenopus laevis oocytes (34 mM) with radiotracers (7), whereas with the same model, the determined Km of MCT1 is ~5 mM (4). However, with a decreased lactate uptake at 1 and 10 mM in SV of Zucker rats, it seems that the MCT1 content of our vesicles was sufficient to detect visible changes in lactate transport at physiological concentrations.
From the study of McCullagh et al. (29), it appears that muscle MCT1 content is highly correlated with muscle lactate uptake as well as oxidative capacity. On the other hand, because MCT4 expression was found to be highly correlated with type II fiber type, it has been suggested that this expression may reflect the muscle requirement for lactate efflux (37). This situation is quite different from what occurs in vitro, with predominant MCT4 content in SV and the two isoforms showing identical influx or efflux kinetics (25). Even if basal lactatemia is increased in both rat (present study) and human insulin resistance-related obesity (6), the values do not exceed the value of MCT1 Km, suggesting that, in vivo, MCT1 is still the major isoform for lactate uptake. Interestingly, mixed muscle such as RTA or red gastrocnemius (RG) has a greater lactate uptake than more oxidative muscle (such as SOL) in normal rats (29). Thus the hyperlactatemia in obesity could be more related to a decreased muscle lactate uptake, primarily in mixed muscle where MCT1 content is significantly decreased. We may therefore hypothesize that the decreased MCT1 content in mixed muscles would be sufficient to significantly decrease lactate uptake in vivo at the physiological concentrations found in obesity.
From the recent literature, we know that both MCT1 expression and MCT4 expression are regulated by contractile activity. Endurance training programs and chronic electrical stimulation upregulate MCT1 but not MCT4 (2). On the other hand, denervation (37) and hypokinesia (hindlimb suspension) (11) decrease SV lactate transport as well as MCT1 and MCT4 expression (37). Interestingly, a series of data shows the decreased spontaneous activity in obese compared with lean Zucker rats (12, 16, 36). Indeed, when fed ad libitum, obese Zucker rats appear to be less active than lean ones (16). We can hypothesize that the long-term adaptation in Zucker rats, i.e., chronic hypokinesia, might mimic what occurs in hindlimb suspension for the expression of both MCT isoforms and sarcolemmal lactate transport. Hypokinesia is generally accompanied by reduced skeletal muscle oxidative capacity (11, 38) and increased glycolytic enzymes (28). Here, what might corroborate the published data indicating a decreased oxidative capacity of skeletal muscles from obese Zucker rats is the redistribution in LDH isozymes toward more type M-LDH. LDH-1 was decreased in RTA and EDL (by 70 and 53%, respectively), where oxidative profile is representative of the majority of hindlimb muscles, but it remained unchanged in highly oxidative or highly glycolytic muscles (SOL and WTA). This shift toward type M-LDH could contribute to the decreased lactate oxidation by skeletal muscles, along with reduced oxidative capacity, in obese rats. From the study of McCullagh et al. (29), we know that MCT1 is strongly correlated with lactate uptake, oxidative capacity, and type H-LDH isozymes. Because the Zucker rats had fiber type-dependent alterations in MCT1 expression and LDH isozymes, we reevaluated the validity of the correlations between MCT1 and LDH isozymes in this model. MCT1 content in control rats was correlated with LDH-3 (r = 0.95) but not with the LDH-2 isozyme, whereas in Zucker rats it was correlated with both the LDH-2 and LDH-3 isozymes (r = 0.96 and 0.98). A correlation between MCT1 content and the LDH-5 isozyme was more surprising, because MCT1 content appeared to decrease in the RTA and EDL muscles of Zucker rats only. However, neurons in which LDH-1 is the predominant isozyme do not express MCT1 but rather the MCT2 isoform, whereas glial cells that contain LDH-5 express only the MCT1 isoform (3). As a result, the hypothesis advanced by McCullagh et al. (30) of coordinated regulation of MCT1 and H-LDH subunit genes is not verified, and more complex mechanisms in their expression may be involved.
In summary, we found a decreased total lactate influx in the skeletal muscles of obese Zucker rats by use of the SV model. This decreased uptake of lactate was accompanied by reduction in the skeletal muscle expression of the MCT1 and MCT4 isoforms, both of which appeared fiber type dependent, with a more pronounced decrease in MCT4 expression. As a consequence of the model, MCT4 content was also reduced in SV of the Zucker rats, which could explain the decreased maximal rate of lactate uptake. We hypothesized that the reduced spontaneous activity of the strain would account for the decreased expression of both MCT isoforms. Consistent with this hypothesis is the redistribution in LDH isozymes in the skeletal muscle of the Zucker rats, which also appeared fiber type specific. A strong relationship was found between LDH-5 and MCT1 only in the obese rats. Although the SV model may be not strictly representative of what occurs in vivo, our evidence suggests that the decreased lactate uptake, along with a shift toward type M-LDH, could reduce skeletal muscle lactate oxidation in obese Zucker rats and thus contribute to basal hyperlactatemia. Further investigations are needed, however, to evaluate the relative part of the impaired skeletal muscle metabolism and exchanges in obesity-related hyperlactatemia. As we pointed out in the introductory paragraphs, adipose mass appears to be a significant site of lactate production. Whether impaired lactate transport and metabolism of other organs, including adipose tissue, participate in obesity-impaired lactate clearance remains to be established.
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ACKNOWLEDGEMENTS |
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We thank G. A. Brooks, Department of Human Dynamics and Integrative Biology, University of California, Berkeley, for the generous gift of MCT1 and MCT4 anti-rat antibodies and synthetic peptides. We also thank M. Rossi, Service de Médecine Nucléaire, CHU de Montpellier, for advice and assistance regarding the handling of the radiochemical materials.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Py, Département de Physiologie, Laboratoire de Physiologie des Interactions, Institut de Biologie, Boulevard Henri IV, 34060 Montpellier, France (E-mail: py{at}medecine.univ-montp1.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 19 June 2001; accepted in final form 7 August 2001.
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