1 Departments of Surgery,
2 Neurology, and
3 Molecular and Cellular
Physiology, Epinephrine and amylin stimulate glycogenolysis,
glycolysis, and
Na+-K+-ATPase
activity in skeletal muscle. However, it is not known whether these
hormones stimulate glycolytic ATP production that is specifically
coupled to ATP consumption by the
Na+-K+
pump. These studies correlated glycolysis with
Na+-K+-ATPase
activity in resting rat extensor digitorum longus and soleus muscles
incubated at 30°C in well-oxygenated medium. Lactate production
rose three- to fourfold, and the intracellular
Na+-to-K+
ratio
(Na+/K+)
fell with increasing concentrations of epinephrine or amylin. In
muscles exposed to epinephrine at high concentrations (5 × 10
ouabain; lactate; oxygen consumption; metabolic compartmentation
EXTREME ELEVATION OF LACTIC ACID in the blood is
observed after major trauma or during generalized sepsis. Traditionally
this hyperlactacidemia has been interpreted as anerobic glycolysis in
skeletal muscle and therefore attributed to either poor tissue perfusion and reduced O2 delivery
or mitochondrial dysfunction (31). In many cases, however, tissue
hypoperfusion has not been demonstrated, nor can the elevated blood
lactate be corrected by therapies designed to improve
O2 delivery to tissues. Severe injury also results in extreme elevation of epinephrine in blood. The
blood concentration of lactic acid rises after administration of either
epinephrine or the peptide hormone amylin (51). Each of these hormones
binds to specific receptors on skeletal muscle, resulting in increased
activity of glycogen phosphorylase, increased glycogen breakdown, and
increased aerobic glycolysis and lactate production (50). Both
epinephrine and amylin also rapidly stimulate the activity of the
Na+-K+
pump in skeletal muscle, thereby leading to a decreased intracellular Na+-to-K+
ratio
(Na+/K+)
and membrane hyperpolarization (9, 37). The effects in skeletal muscle
of these hormones on carbohydrate metabolism, on one hand, and on
activity of the
Na+-K+-ATPase,
on the other, were described separately. Although no relationship has
been suggested between these two classes of effects, previous research
in a variety of systems supports such a linkage.
Recent studies from our laboratories (22) showed that aerobic
glycolysis was stimulated in rat skeletal muscles in vitro by exposure
to the Na+ ionophore monensin,
which stimulates
Na+-K+-ATPase
activity by raising the intracellular concentration of Na+
([Na+]i).
In that study, ouabain inhibited monensin-stimulated lactate production, suggesting that increased activity of the
Na+-K+
pump resulted in increased aerobic glycolysis. In these studies, ouabain also reduced the increased lactate production in vitro by
muscles from septic rats, suggesting that increased muscle lactate
production in sepsis may be related to increased
Na+-K+-ATPase
activity rather than to anerobic metabolism.
The aim of the present studies was to further validate the hypothesis
that increased
Na+-K+-ATPase
activity in skeletal muscle can result in increased aerobic glycolysis.
Therefore, we examined the effects of epinephrine or amylin on activity
of the
Na+-K+
pump and on aerobic glycolysis in skeletal muscle. A number of interrelated issues were addressed:
1) Is lactate production that is
stimulated by epinephrine or amylin correlated with increased activity
of the
Na+-K+
pump? 2) Can epinephrine- or
amylin-stimulated lactate production be inhibited by treatments that
inhibit
Na+-K+
pump activity? 3) Did the incubation
conditions employed in these studies reduce muscle content of
high-energy phosphates? 4) Is glycogenolysis in the absence or presence of epinephrine affected by
ouabain? Our results indicate that, in resting muscle, stimulation of
glycolysis by epinephrine or amylin is primarily the result of
stimulation of the
Na+-K+-ATPase
without concomitant stimulation of oxidative metabolism.
Muscle Incubations
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
7 and 5 × 10
6 M), ouabain
significantly inhibited glycolysis by ~70% in either muscle and
inhibited glycogenolysis by ~40 and ~75% in extensor digitorum
longus and soleus, respectively. In the absence of ouabain, but not in
its presence, statistically significant inverse correlations were
observed between lactate production and intracellular
Na+/K+
for each hormone. Epinephrine had no significant effect on oxygen consumption or ATP content in either muscle. These results suggest for
the first time that stimulation of glycolysis and glycogenolysis in
resting skeletal muscle by epinephrine or amylin is closely linked to
stimulation of active
Na+-K+ transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
In experiments assessing epinephrine and amylin dose-response relationships, one of the pair of EDL or soleus muscles from each animal was exposed to hormone, while the contralateral muscle served as a control and was incubated without hormone. If ouabain was absent, neither muscle was exposed to ouabain, and if ouabain was present, both muscles were exposed to ouabain. After the 1-h incubations, the medium was frozen for assay of lactate production. In experiments in which the time course of lactate accumulation in the medium was followed, 0.33-ml samples of medium were removed from the incubation flasks at 15-min intervals, and the flasks were regassed before being returned to the water bath.
To study the effect of epinephrine in
K+-free medium, KCl and
KH2PO4
were replaced isosmotically with NaCl and
NaH2PO4.
In these studies, one of the pair of muscles was preincubated in normal
Krebs-Henseleit buffer and the other in
K+-free medium. Muscles were then
transferred to fresh medium with the same ionic composition and
containing epinephrine (5 × 106 M) for a 30-min
incubation. The preincubation volume was 5 ml, and preincubation time
was 45 min. These modifications in the incubation conditions were
designed to achieve a more thorough washout of extracellular
K+ during preincubation and to
minimize the rise in medium K+
concentration due to leakage from the muscle during exposure to
epinephrine. It is recognized, however, that these procedures are
unlikely to eliminate completely all
K+ from the region immediately
adjacent to the outer surface of the cell membrane.
Lactate Assay
Lactate production was determined by measuring lactate concentration in incubation medium. Lactate was assayed by a standard microfluorometric enzymatic procedure (27) involving reduction of NAD by lactate dehydrogenase (LDH) to produce NADH, which was detected fluorometrically (excitation: 360 nm, emission: 530 nm) by use of a microplate reader. With the use of 96-well plates (no. 3632, Corning Costar, Cambridge, MA), 50 µl of incubation medium or lactate standards (in triplicate) were pipetted into each well. Then 200 µl of a solution of LDH and NAD in hydrazine-glycine buffer, pH 9.1, were added, followed by agitation (5 min) on a rotary plate-mixer. Forty-five minutes later, fluorescence was measured. Each plate was read three times over a period of ~2 min, and the fluorescence estimation for each sample was thus the average of three readings for triplicate samples.Intracellular Na+ and K+ Contents
For the measurement of intracellular Na+ and K+, washout of ions from the extracellular space was accomplished by a series of four 15-min incubations at 0°C in ~3 ml of Na+- and K+-free buffer containing (in mM) 263 sucrose and 10 Tris, adjusted to pH 7.4 with HCl (10). The series of washouts was begun immediately after the 1-h incubations described in Muscle Incubations. Muscles were then homogenized in 1 ml of 0.4 N trichloroacetic acid with a sonicator (VC 50, Sonics & Materials, Danbury, CT), followed by centrifugation to remove precipitated proteins. Na+ and K+ concentrations were measured in the supernate, after appropriate dilution with deionized water, with a Shimadzu AA-6200 atomic absorption spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD) operated in emission mode. To correct for loss of Na+ during washout, intracellular Na+ was multiplied by 1.46 as previously described (10). Previous studies have shown that no K+ is lost during this washout procedure (17). The fraction of wet weight that comprised the extracellular water was assumed not to change significantly over the various treatments and, therefore, intracellular Na+ and K+ contents were expressed as micromoles per gram wet weight (17). The calculated intracellular Na+/K+ does not depend on absolute concentrations in intracellular water.Resting O2 Consumption
Animals were anesthetized with pentobarbital sodium (~60 mg/kg), and the EDL and soleus muscles were exposed. The resting length of each muscle was measured in situ. Muscles were then removed with tendons intact and were maintained at resting length during the experiments. Dissected muscles were suspended vertically in a sealed chamber (volumeResting O2 consumption was
determined before and after epinephrine treatment. Chamber
PO2 was
recorded for 30 min with the muscle in place, and an aliquot of chamber
fluid was then taken and frozen immediately for lactate analysis. The
chamber was flushed thoroughly with fresh oxygenated buffer, and the
muscle was allowed to equilibrate for 30 min. Epinephrine was injected
into the chamber (final concentration = 5 × 106 M), and
O2 consumption was followed for 30 min. A second aliquot of chamber fluid was then taken for lactate
analysis. Rates of O2 consumption
were calculated using Acknowledge data acquisition software (Biopac
Systems, Santa Barbara, CA). Pre- and postepinephrine O2 consumption was normalized to
muscle mass and expressed as a function of time
(nmol · g
1 · min
1).
Muscle Content of ATP, Creatine Phosphate, and Glycogen
ATP and creatine phosphate were assayed using an HPLC procedure (47) modified as follows. At the end of incubations, muscles were frozen rapidly in liquid N2. Frozen muscles were placed into custom-fabricated plastic (Kel-F) tubes containing 0.1 ml of 0.1 N HCl in methanol (precooled to liquid N2 temperature,The HPLC instrument (Waters, Milford, MA) consisted of an autosampler (model 600E), absorbance monitor (model 490E), gradient-mixing pump, computer, and software (Millennium) for instrument control, data collection, and analysis. Separation was performed on a 4.6 mm × 100 mm SynChropak AX100 anion-exchange column (Micra Scientific, Lafayette, IN) operated at room temperature. Creatine phosphate, ADP, and ATP were well separated at a flow rate of 2 ml/min by use of a gradient of two buffers, buffer A (50 mM KH2PO4, pH 4.5) and buffer B (750 mM KH2PO4, titrated to pH 2.7 with 85% phosphoric acid), as follows: isocratic 100% buffer A for 2 min, a linear gradient to 58% buffer B from 2 min to 10 min, a linear gradient to 100% buffer B from 10 min to 11 min, and isocratic 100% buffer B from 11 min to 16 min. Eluted compounds were detected by absorbance at 210 nm and quantitated by comparison with standards of known concentration.
Chemicals
All chemicals purchased were of analytic grade. Rat amylin-(1Statistical Analysis of Results
Statistical analysis was performed using the programs SAS (SAS Institute, Cary, NC) and Stata (Stata, College Station, TX). Significance of differences between groups was determined using the Student-Newman-Keuls test after two-way repeated-measures ANOVA or Student's t-test for paired or unpaired observations, as appropriate. Differences were considered significant at P < 0.05. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of Epinephrine and Amylin on Lactate Production and Intracellular Na+ and K+ in the Absence of Ouabain
Epinephrine.
In both EDL and soleus, epinephrine significantly increased lactate
production in a dose-dependent manner, with the maximum stimulation
being ~400% of control values (Table 1).
[Na+]i
and
Na+/K+
were measured to assess the activity of the
Na+-K+-ATPase.
In EDL, compared with untreated controls, epinephrine significantly
decreased mean
Na+/K+
at all concentrations tested. In EDL, at all concentrations except the
lowest tested (5 × 109 M), epinephrine
significantly decreased
[Na+]i
(Table 1). In the soleus, epinephrine at all concentrations except 5 × 10
9 M significantly
decreased
[Na+]i
and
Na+/K+.
The intracellular concentration of
K+
([K+]i)
was significantly increased by epinephrine in EDL when all epinephrine
concentrations were considered together [control vs. epinephrine:
80.6 ± 1.7 vs. 85.3 ± 1.0 µmol/g, means ± SE
(n = 33),
P < 0.05 by paired
t-test], but not when any one
concentration was considered alone. No significant effect of
epinephrine was found on
[K+]i
of soleus (data not shown).
|
Amylin.
Amylin also stimulated lactate production (Table
2). Maximum stimulation of lactate
production by amylin appeared to be somewhat less than that by
epinephrine. In EDL, at all concentrations tested, amylin significantly
lowered
[Na+]i
and, at all concentrations except the lowest
(109 M), significantly
decreased
Na+/K+
(Table 2). In soleus, amylin significantly decreased
[Na+]i
and
Na+/K+
at all concentrations tested except
10
9 M. No significant
effect of amylin was found on
[K+]i
of EDL or of soleus (data not shown).
|
Effects of Ouabain on Lactate Production and Intracellular Na+ and K+
Ouabain treatment was associated with a significant decrease in lactate production in control muscles (not exposed to epinephrine or amylin). In control muscles, ouabain significantly reduced lactate production in both EDL [no ouabain (n = 70) vs. ouabain (n = 56): 4.6 ± 0.1 vs. 2.2 ± 0.1 µmol · gEpinephrine.
When ouabain was present, epinephrine significantly stimulated lactate
production, but this stimulation was much less in the presence of
ouabain than in its absence (Table 1). This effect of ouabain is
readily apparent when the difference in lactate production between
epinephrine-treated and control muscles of each pair is plotted as a
function of epinephrine concentration (Fig.
1); it can be estimated that ouabain
inhibited the effect of epinephrine on lactate production by ~80%.
|
Amylin.
When ouabain was present, amylin significantly stimulated lactate
production, but this stimulation was much less in the presence of
ouabain than in its absence (Table 2). In the presence of ouabain,
amylin appeared to be less effective than epinephrine in stimulating
glycolysis. When this difference is taken into account, the maximal
ouabain-inhibitable lactate production that was stimulated by
epinephrine was similar to that stimulated by amylin (EDL 14-15
µmol · g1 · h
1,
soleus 10-12
µmol · g
1 · h
1).
In ouabain-treated muscles, amylin exposure at
10
6 M resulted in
significantly lower
[Na+]i
only in EDL muscle, whereas
Na+/K+
was not significantly altered by amylin in either muscle.
Linearity of Lactate Production
To determine whether lactate production was proportional to incubation time in the presence or absence of epinephrine (5 × 10
|
Correlation of Effects of Epinephrine and Ouabain on Lactate Production and Na+/K+
Inspection of Tables 1 and 2 suggests that a significant correlation may be demonstrated in both EDL and soleus between lactate production and Na+/K+ in the presence of hormones and in the absence of ouabain. Linear regression analysis was performed for EDL and soleus, with consideration of data from either epinephrine (data from Table 1) or amylin (data from Table 2) studies separately or of combined data from studies with both hormones. Statistically significant inverse relationships were observed between lactate production rate and Na+/K+ when only the epinephrine-treated EDL (P = 0.0001, n = 33) and soleus (P < 0.0001, n = 33) were considered. Statistically significant inverse relationships were also found between lactate production rate and Na+/K+ when only the amylin-treated EDL (P = 0.02, n = 28) and soleus (P < 0.0001, n = 28) were considered. The observed relationships (Fig. 3) between lactate production and Na+/K+ (combined data from epinephrine-treated and amylin-treated muscles) suggest that the slope of regression lines was markedly steeper in EDL than in soleus because 1) resting Na+/K+ was lower in EDL than in soleus, 2) epinephrine and amylin treatments reduced Na+/K+ to similar levels in both muscles, and 3) lactate production was stimulated somewhat more in EDL than in soleus. No significant correlations were obtained between these two factors in untreated muscles (Fig. 3, inset) or in muscles incubated in the presence of ouabain (Fig. 3, main and inset graphs).
|
Effect of Incubation in K+-Free Medium on Lactate Production and Na+/K+
To assess whether the ouabain-induced decrease in glycolysis was related to inhibition of the Na+-K+-ATPase rather than to some unknown effect of ouabain on glucose metabolism, muscles were exposed to epinephrine (5 × 10
|
Effect of Epinephrine on O2 Consumption Rate by EDL and Soleus Muscles
Rates of O2 consumption by EDL and soleus muscles were measured in normal Krebs-Henseleit medium with 10 mM glucose both before and after addition of epinephrine (5 × 10
|
Effect of Ouabain and Epinephrine on Muscle Contents of Creatine Phosphate, ATP, and Glycogen
To assess whether stimulation of glycolysis by epinephrine might be caused by impairment of ATP production or depletion of high-energy phosphates, we measured muscle content of creatine phosphate and ATP in muscles incubated for 2 h in the absence or presence of ouabain and of epinephrine (5 × 10Differences between EDL and Soleus Muscles in the Absence of Hormones or Ouabain
When only control muscles (not exposed to epinephrine or amylin) incubated in the absence of ouabain are considered, resting lactate production was significantly higher in EDL than in soleus [EDL (n = 70) vs. soleus (n = 71): 4.6 ± 0.1 vs. 3.4 ± 0.1 µmol · g ![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Epinephrine plays well-defined roles, not only in regulation of fat and carbohydrate metabolism but also in control of contractile function in smooth, cardiac, and skeletal muscle. Amylin is a recently discovered peptide hormone that is cosecreted with insulin from the pancreas. Although amylin's specific functions are uncertain, the hormone may contribute to control of whole body glucose disposition by modulating glycogen metabolism in skeletal muscle (50). Intravenous epinephrine administration increases the circulating lactate concentration (5, 38). A major source of this lactate is assumed to be glycolysis in skeletal muscle, for which all or most of the glucose is thought to be derived from muscle glycogen breakdown (3). Glycogenolysis in skeletal muscle cannot directly support the circulating glucose concentration because muscle glucose-6-phosphatase activity is very low (19). Stimulation of muscle glycolysis by epinephrine is traditionally attributed to increased activities of the enzymes glycogen phosphorylase and phosphofructokinase (PFK), with the latter considered to catalyze the flux-controlling reaction in glycolysis (6, 13). Amylin, also, stimulates glycogenolysis and glycolysis and inhibits glycogen synthesis in rat muscle (4, 52).
Stimulation by epinephrine of the
Na+-K+
pump in skeletal muscle has been described previously. After treatment
with isoproterenol, guinea pig EDL exhibited membrane hyperpolarization
and increased twitch tension; these responses were inhibited by the
-receptor antagonist propranolol, by ouabain, or by reduced
extracellular K+ concentration
(42). In rat soleus, isoproterenol or epinephrine decreased muscle
Na+/K+,
increased both 22Na efflux and
42K influx, and caused membrane
hyperpolarization, effects that could be inhibited by either ouabain or
propranolol (11). The effects of isoproterenol or epinephrine on ion
transport or membrane potential could be mimicked by a combination of
dibutyryl cAMP and theophylline. These effects could be inhibited by
ouabain as well, indicating that epinephrine stimulated the
Na+-K+-ATPase
via increased intracellular concentration of cAMP (11). It has been
suggested that these effects of epinephrine counteract the dissipation
in transmembrane Na+ and
K+ gradients caused by repetitive
muscle excitation and, therefore, are important for maintaining
contractile performance and for increasing endurance during sustained
exercise (20, 32).
Thus, with regard to skeletal muscle, epinephrine's actions are at least threefold: 1) to stimulate glycolysis, 2) to increase release of gluconeogenic precursors, and 3) to modify contractile function by maintaining or increasing transmembrane gradients for Na+ and K+. The present studies suggest that, at least in resting muscle, epinephrine accomplishes these diverse functions largely through a single mechanism, stimulation of the Na+-K+-ATPase. Moreover, these studies suggest that this mechanism can account also for the effects of amylin on skeletal muscle. This conclusion is supported by two general observations. First, both the rate of lactate production and that of Na+-K+ transport, as indicated by Na+/K+, increased in proportion to the concentration of hormones added to the medium (Table 1). Moreover, lactate production was inversely correlated with Na+/K+ in both EDL and soleus over the tested range of epinephrine or amylin concentrations (Fig. 3). Second, inhibition of the Na+-K+ pump, either by ouabain or by K+ deficiency, inhibited both lactate production and Na+-K+ transport.
Linkage between glycolysis and activity of the Na+-K+ pump was demonstrated in early studies in rat diaphragm, in which lactate production was reduced either by ouabain or by incubation in K+-free medium, treatments that inhibit the Na+-K+-ATPase (7, 8). Stimulation of both glycogenolysis and lactate production in diaphragm by epinephrine could be partially inhibited by ouabain or digitoxin (25). A close relationship between aerobic glycolysis and Na+-K+-ATPase activity has been found in a variety of other cell types, including not only erythrocytes but also Ehrlich ascites tumor cells, neurons, glia, and vascular smooth muscle (16, 26, 33, 34, 36, 39, 46), suggesting that coupling of glycolysis to energy utilization by membrane transport processes may be a general phenomenon (21). If so, then stimulation of skeletal muscle glycolysis by epinephrine or amylin may also be related to increased activity of the Na+-K+-ATPase.
The present studies confirm that epinephrine and amylin cause a marked
dose-dependent stimulation of lactate production by skeletal muscle
(Tables 1 and 2). Stimulation of lactate production by epinephrine
appeared to reach a maximum at 5 × 106 M, the highest
concentration tested (Table 1, Fig. 1). In the present studies,
epinephrine increased the rate of lactate production to a much greater
degree (~400% of control) than that previously observed (25) in the
diaphragm (~15-25%). This difference is likely due to the high
rate of lactate production by control diaphragm portions incubated in
the absence of epinephrine. Pieces of diaphragm may have severed muscle
fibers, allowing influx of Na+
from the medium and causing an increase in
[Na+]i.
Treatments that raise
[Na+]i
have been shown to stimulate
Na+-K+-ATPase
activity and aerobic glycolysis (16, 22). Moreover, the diaphragm
portions were incubated at 37°C in an atmosphere of 20%
O2 (air), conditions which may
provide inadequate oxygenation and stimulate anerobic glycolysis.
Because the present studies employed intact, well-oxygenated skeletal
muscles, the observed stimulation of lactate production by epinephrine
is probably more representative of conditions in vivo.
The present studies also confirm that epinephrine lowers [Na+]i and muscle Na+/K+ through stimulation of the Na+-K+-ATPase. Thus, in both EDL and soleus, Na+/K+ fell in proportion to the epinephrine concentration, and this effect was inhibited by ouabain (Table 1). Similar results were obtained for amylin (Table 2), thereby confirming and extending previous observations (9). Ouabain did not completely inhibit the stimulation of lactate production by epinephrine (Fig. 2). This ouabain-resistant increase in glycolysis may reflect ATP consumption by processes other than Na+-K+ pumping that are stimulated by epinephrine or amylin. On the other hand, the ouabain-resistant lactate production may simply reflect increased glycogenolysis, and thus increased availability of glucose-6-phosphate, for glycolysis in the cell generally. The concentration of ouabain (1 mM) used in these studies has been reported to result in a maximal inhibition of the Na+-K+-ATPase in rat skeletal muscles (11). However, even in the presence of ouabain, epinephrine treatment was associated with a significant reduction in [Na+]i of soleus muscles (Table 1), suggesting that inhibition of the Na+-K+-ATPase by ouabain was not complete. If all Na+-K+ pump activity was not inhibited by ouabain in the present studies, then the fraction of epinephrine-stimulated glycolysis that is coupled to Na+-K+-ATPase activity would be higher than the 80% estimated from Fig. 1.
The present results clearly suggest that, in resting skeletal muscle,
the stimulation of glycolysis by epinephrine or amylin is closely
linked to stimulation of the
Na+-K+-ATPase.
This conclusion is supported by two observations. The first is the
concurrent inhibition of lactate production and
Na+-K+
pump activity by either ouabain or incubation in
K+-free medium. The second
observation is the significant inverse correlation between the rate of
lactate production and
Na+/K+.
Together, these observations suggest that both epinephrine and amylin
stimulate ATP consumption by the
Na+-K+-ATPase
that is coupled to ATP production by glycolysis and, therefore, that
the rate of aerobic lactate production by resting skeletal muscle in
the presence of these hormones provides a rough index of
Na+-K+
pump activity. Consistent with this suggestion, epinephrine
(106 M) was previously
shown to raise K+ uptake by ~0.2
µmol · g
1 · min
1
(11) in rat soleus muscles incubated under conditions similar to those
in the present studies. If we assume a cost of 1 ATP per 2 K+ ions taken up, this increase in
K+ uptake would require an
increase in ATP consumption of 0.1 µmol · g
1 · min
1
or 6 µmol · g
1 · h
1.
In the present studies, the maximal increase in ouabain-suppressible lactate production, corresponding to production of an equimolar amount
of ATP, was ~10
µmol · g
1 · h
1,
in reasonable agreement with the previously reported increase in
K+ uptake.
The observed correlation between lactate production and intracellular
Na+/K+
deserves comment. More appropriately, the rate of lactate production should have been correlated to the rate of active
Na+-K+
pumping. It was previously reported that epinephrine (6 × 106 M) reduced
[Na+]i
by ~50% within 10-15 min and only by a further ~20%
thereafter, up to 90 min (11). This observation might indicate that the highest rate of the
Na+-K+
pumping occurred soon after exposure to epinephrine. Therefore we
examined whether the rate of lactate production was higher during the
early times after exposure to epinephrine (Fig. 2). To the contrary,
lactate production was approximately linear over the four 15-min
intervals and, during the 2-h incubations (Table 3), the calculated
hourly rate of lactate production was close to that in the 1-h
incubations. Thus, even though most of the change in
[Na+]i
may have occurred within 15 min of exposure to epinephrine, the rate of
lactate production remained elevated for up to 2 h. This observation
suggests that a steady, higher rate of
Na+-K+
pumping is required to maintain the lower steady-state
Na+/K+.
One explanation for this may be more rapid inward leakage of Na+ in the presence of epinephrine
due to the greater transmembrane Na+ gradient and more polarized
membrane potential (11).
The present results suggest that the stimulation of glycogenolysis and
that of the
Na+-K+-ATPase
by epinephrine, both effects being the consequences of increased
intracellular cAMP, are coordinated effects. Epinephrine (5 × 106 M) caused a
statistically significant reduction in muscle glycogen in both EDL and
soleus, and this effect was significantly inhibited by ouabain (Table
3). This observation suggests that inhibition of the
Na+-K+-ATPase,
thereby slowing the rate of ATP utilization, also inhibited glycogenolysis, perhaps by resulting in high levels of glucose 6-phosphate. Enzymatic control of glycogen metabolism is complex but
may be strongly influenced by glucose 6-phosphate concentration (41).
In the earlier diaphragm studies, epinephrine
(~10
5 M) lowered tissue
glycogen content, whereas ouabain treatment (10
4 M) resulted in higher
tissue glycogen content, either in the absence or in the presence of
epinephrine (25). Moreover, in epinephrine-treated diaphragm, ouabain
resulted in significant increases in the content of glucose 6-phosphate
and fructose 6-phosphate and a significant decrease in that of fructose
1,6-bisphosphate. These results suggest that ouabain treatment resulted
in inhibition of PFK. Because ouabain is not known to have any direct
effect on PFK activity, it is reasonable to suggest that ouabain
exerted its effect on PFK by decreasing ATP utilization by the
Na+-K+-ATPase,
thereby decreasing the availability of ADP and increasing that of ATP.
PFK is inhibited by ATP and stimulated by ADP and inorganic phosphate.
The present observations thus run counter to traditional assumptions,
which attribute primary control of glycolytic flux to activities of
glycogenolytic and glycolytic enzymes, and they suggest that the rate
of ATP utilization by the
Na+-K+-ATPase
can regulate the rates of aerobic glycolysis and glycogen breakdown.
The effect of epinephrine on muscle O2 consumption was examined because it has been reported that epinephrine raises O2 consumption in the perfused rat hindquarter preparation, both at rest and during electrical stimulation of the sciatic nerve (38). If epinephrine were found to stimulate an increase in O2 consumption in muscle under the conditions of the present studies, then it might be concluded that epinephrine stimulated multiple energy-requiring processes and, thus, a broad stimulation of ATP turnover. Increased glycolysis would not be surprising under conditions of a nonspecific increase in metabolism. However, epinephrine did not significantly affect O2 consumption in the present studies (Table 3). Failure of epinephrine to stimulate O2 consumption in these resting muscles suggests that epinephrine's stimulation of glycolysis was specific and not contingent on a general acceleration of muscle metabolism. Epinephrine may increase O2 consumption in the perfused hindlimb by stimulating lipolysis and raising the concentration of free fatty acids in the recirculating perfusate (15).
There was no evidence that epinephrine treatment stimulated glycolysis by inhibiting oxidative ATP production and thereby reducing high-energy phosphate stores. Moreover, there was no evidence that ouabain inhibited glycolysis by counteracting such an effect. The present values for creatine phosphate and ATP, measured after a 2-h incubation either in the absence or the presence of epinephrine (Table 3), were in good agreement with those previously published for rat EDL and soleus (24). Finally, incubation in K+-free medium, in which the Na+-K+-ATPase was partially inhibited without the use of ouabain, also resulted in a parallel inhibition of epinephrine-stimulated lactate production. The similarity between the effects of ouabain and K+-free medium strongly suggests that both treatments affected glycolysis and Na+-K+ pump activity concurrently, because the rate of glycolysis was indirectly regulated by Na+-K+-ATPase activity.
This relationship between aerobic glycolysis and activity of the
Na+-K+-ATPase
is also supported by the observations in control muscles not exposed to
hormones. The present studies confirmed previous reports (17) that the
resting
[Na+]i
and
Na+/K+
are significantly lower in EDL than in soleus. The total number of
[3H]ouabain binding
sites, a measure of number of active
Na+-K+
pumps, has consistently been shown to be higher in resting EDL than in
resting soleus (17, 23). These observations suggest that the difference
in resting
[Na+]i
and
Na+/K+
in EDL and soleus is determined by the number of active
Na+-K+
pumps present in these two muscles. In the present studies, both the
total lactate production and the ouabain-inhibitable lactate production
were significantly higher in resting EDL than in resting soleus. In
contrast, a previous study from this laboratory found no significant
difference between EDL and soleus muscles in resting lactate production
(see Table 1 of Ref. 22). Possible reasons for this discrepancy are
that the earlier data were obtained from fewer muscles and displayed
greater variability. When the previous data were combined with the
present data, basal lactate production by EDL was still
significantly greater than that by soleus [EDL (n = 129) vs. soleus
(n = 121): 4.0 ± 0.1 vs. 3.6 ± 0.1 µmol · g1 · h
1,
means ± SE, P < 0.05].
Therefore the rate of aerobic glycolysis may provide an index of
Na+-K+-ATPase
activity in untreated resting muscles also.
Coupling between glycolysis and Na+-K+-ATPase activity was examined in detail in early studies in erythrocytes (33, 45, 46). Lactate production by erythrocytes is stimulated by increased [Na+]i and inhibited by ouabain (45). Preparations of erythrocyte membranes exhibit glycolytic enzyme activities (40) as well as Na+-K+-ATPase activity. Na+ transport by isolated membranes was supported either by added ATP or by substrates for glyceraldehyde-3-phosphate dehydrogenase or phosphoglycerate kinase (30). Such studies suggested that ATP produced by glycolysis might be transferred within the microenvironment of the cell membrane to the Na+-K+-ATPase (30). Transfer of ATP within a restricted environment associated with the sarcolemmal membrane may account for the apparent coupling between Na+-K+-ATPase activity and glycolytic rate observed in the present studies.
Metabolic compartmentation was demonstrated by Lynch and Paul (28, 29) in smooth muscle when they showed that glycogen-derived glucose is metabolized in one compartment to CO2 and H2O, while, in another, extracellular glucose is metabolized to lactate. Using isolated plasma membrane vesicles, Paul et al. (35) further demonstrated both a functionally complete glycolytic enzyme cascade and ATP-dependent calcium pumping, suggesting an association among the membrane, calcium pumps, and glycolytic enzymes. These studies were later confirmed by others (48, 49). Studies in isolated working hearts have also provided evidence of metabolic compartmentation and suggest preferential utilization of glycolytically derived ATP by the Na+-K+-ATPase (14, 44). Taken together, these results suggest that coupling of an ATP-consuming membrane pump to an ATP-generating glycolytic cascade may occur frequently. The possibility that glycolytic and oxidative compartments may function independently within cells may be of great value in the clinical interpretation and treatment of symptoms of burn injury, sepsis, or hemorrhage. The existence in skeletal muscle of a linkage among epinephrine, the Na+-K+-ATPase, glycogenolysis, and aerobic glycolysis might explain why the increased lactate production of these pathological states is unresponsive to increased O2 delivery.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by Grant 8630 from Shriners Hospitals for Children and National Institute of General Medical Sciences Grant GM-54775.
![]() |
FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: J. H. James, Univ. of Cincinnati Medical Center, Dept. of Surgery/ Mail Location 0558, 231 Bethesda Ave., Cincinnati, OH 45267-0558 (E-mail: jamesjh{at}ucmail.uc.edu).
Received 3 December 1998; accepted in final form 25 March 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Balasubramaniam, A.,
V. Renugopalakrishnan,
M. Stein,
J. E. Fischer,
and
W. T. Chance.
Syntheses, structures and anorectic effects of human and rat amylin.
Peptides
12:
919-924,
1991[Medline].
2.
Bonen, A.,
M. G. Clark,
and
E. J. Henriksen.
Experimental approaches in muscle metabolism: hindlimb perfusion and isolated muscle incubations.
Am. J. Physiol.
266 (Endocrinol. Metab. 29):
E1-E16,
1994
3.
Bonen, A.,
J. C. McDermott,
and
C. A. Hutber.
Carbohydrate metabolism in skeletal muscle: an update of current concepts.
Int. J. Sports Med.
10:
385-401,
1989[Medline].
4.
Castle, A. L.,
C.-H. Kuo,
and
J. L. Ivy.
Amylin influences insulin-stimulated glucose metabolism by two independent mechanisms.
Am. J. Physiol.
274 (Endocrinol. Metab. 37):
E6-E12,
1998
5.
Chu, C. A.,
D. K. Sindelar,
D. W. Neal,
E. J. Allen,
E. P. Donahue,
and
A. D. Cherrington.
Comparison of the direct and indirect effects of epinephrine on hepatic glucose production.
J. Clin. Invest.
99:
1044-1056,
1997
6.
Clark, M. G.,
and
G. S. Patten.
Adrenergic control of phosphofructokinase and glycolysis in rat heart.
Curr. Top. Cell. Regul.
23:
127-176,
1984[Medline].
7.
Clausen, T.
The relationship between the transport of glucose and cations across cell membranes in isolated tissues. I. Stimulation of glycogen deposition and inhibition of lactic acid production in diaphragm, induced by ouabain.
Biochim. Biophys. Acta
109:
164-171,
1965[Medline].
8.
Clausen, T.
The relationship between the transport of glucose and cations across cell membranes in isolated tissues. II. Effects of K+-free medium, ouabain and insulin upon the fate of glucose in rat diaphragm.
Biochim. Biophys. Acta
120:
361-368,
1966[Medline].
9.
Clausen, T.
Long- and short-term regulation of the Na+-K+ pump in skeletal muscle.
News Physiol. Sci.
11:
24-30,
1996.
10.
Clausen, T.,
S. L. Andersen,
and
J. A. Flatman.
Na+-K+ pump stimulation elicits recovery of contractility in K+-paralysed rat muscle.
J. Physiol. (Lond.)
472:
521-536,
1993[Abstract].
11.
Clausen, T.,
and
J. A. Flatman.
The effect of catecholamines on Na-K transport and membrane potential in rat soleus muscle.
J. Physiol. (Lond.)
270:
383-414,
1977[Medline].
12.
Crow, M. T.,
and
M. J. Kushmerick.
Chemical energetics of slow- and fast-twitch muscles of the mouse.
J. Gen. Physiol.
79:
147-166,
1982[Abstract].
13.
Dietz, M. R.,
J. L. Chiasson,
T. R. Soderling,
and
J. H. Exton.
Epinephrine regulation of skeletal muscle glycogen metabolism. Studies utilizing the perfused rat hindlimb preparation.
J. Biol. Chem.
255:
2301-2307,
1980
14.
Dizon, J.,
D. Burkhoff,
J. Tauskela,
J. Whang,
P. Cannon,
and
J. Katz.
Metabolic inhibition in the perfused rat heart: evidence for glycolytic requirement for normal sodium homeostasis.
Am. J. Physiol.
274 (Heart Circ. Physiol. 43):
H1082-H1089,
1998
15.
Early, R. J.,
and
S. P. Spielman.
Muscle respiration in rats is influenced by the type and level of dietary fat.
J. Nutr.
125:
1546-1553,
1995[Medline].
16.
Erecinska, M.,
F. Dagani,
D. Nelson,
J. Deas,
and
I. A. Silver.
Relations between intracellular ions and energy metabolism: a study with monensin in synaptosomes, neurons, and C6 glioma cells.
J. Neurosci.
11:
2410-2421,
1991[Abstract].
17.
Everts, M. E.,
and
T. Clausen.
Activation of the Na-K pump by intracellular Na in rat slow- and fast-twitch muscle.
Acta Physiol. Scand.
145:
353-362,
1992[Medline].
18.
Everts, M. E.,
T. Lomo,
and
T. Clausen.
Changes in K+, Na+ and calcium contents during in vivo stimulation of rat skeletal muscle.
Acta Physiol. Scand.
147:
357-368,
1993[Medline].
19.
Gamberucci, A.,
P. Marcolongo,
R. Fulceri,
R. Giunti,
S. L. Watkins,
I. D. Waddell,
A. Burchell,
and
A. Benedetti.
Low levels of glucose-6-phosphate hydrolysis in the sarcoplasmic reticulum of skeletal muscle: involvement of glucose-6-phosphatase.
Mol. Membr. Biol.
13:
103-108,
1996[Medline].
20.
Harrison, A. P.,
O. B. Nielsen,
and
T. Clausen.
Role of Na+-K+ pump and Na+ channel concentrations in the contractility of rat soleus muscle.
Am. J. Physiol.
272 (Regulatory Integrative Comp. Physiol. 41):
R1402-R1408,
1997
21.
Ishida, Y.,
I. Riesinger,
T. Wallimann,
and
R. J. Paul.
Compartmentation of ATP synthesis and utilization in smooth muscle: roles of aerobic glycolysis and creatine kinase.
Mol. Cell Biochem.
133-134:
39-50,
1994.
22.
James, J. H.,
C. H. Fang,
S. J. Schrantz,
P. O. Hasselgren,
R. J. Paul,
and
J. E. Fischer.
Linkage of aerobic glycolysis to sodium-potassium transport in rat skeletal muscle. Implications for increased muscle lactate production in sepsis.
J. Clin. Invest.
98:
2388-2397,
1996
23.
Kjeldsen, K.,
M. E. Everts,
and
T. Clausen.
Effects of semi-starvation and potassium deficiency on the concentration of [3H]ouabain-binding sites and sodium and potassium contents in rat skeletal muscle.
Br. J. Nutr.
56:
519-532,
1986[Medline].
24.
Kushmerick, M. J.,
T. S. Moerland,
and
R. W. Wiseman.
Mammalian skeletal muscle fibers distinguished by contents of phosphocreatine, ATP, and Pi.
Proc. Natl. Acad. Sci. USA
89:
7521-7525,
1992[Abstract].
25.
Kypson, J.,
L. Triner,
and
G. G. Nahas.
The effects of cardiac glycosides and their interaction with catecholamines on glycolysis and glycogenolysis in skeletal muscle.
J. Pharmacol. Exp. Ther.
164:
22-30,
1968[Medline].
26.
Lipton, P.,
and
K. Robacker.
Glycolysis and brain function: [K+]o stimulation of protein synthesis and K+ uptake require glycolysis.
Federation Proc.
42:
2875-2880,
1983[Medline].
27.
Lowry, O. H.,
and
J. V. Passonneau.
A collection of metabolite assays.
In: A Flexible System of Enzymatic Analyses. Orlando, FL: Academic, 1972, p. 199-201.
28.
Lynch, R. M.,
and
R. J. Paul.
Compartmentation of glycolytic and glycogenolytic metabolism in vascular smooth muscle.
Science
222:
1344-1346,
1983[Medline].
29.
Lynch, R. M.,
and
R. J. Paul.
Compartmentation of carbohydrate metabolism in vascular smooth muscle.
Am. J. Physiol.
252 (Cell Physiol. 21):
C328-C334,
1987[Abstract].
30.
Mercer, R. W.,
and
P. B. Dunham.
Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes.
J. Gen. Physiol.
78:
547-568,
1981[Abstract].
31.
Mizock, B. A.
Alterations in carbohydrate metabolism during stress: a review of the literature.
Am. J. Med.
98:
75-84,
1995[Medline].
32.
Overgaard, K.,
O. B. Nielsen,
and
T. Clausen.
Effects of reduced electrochemical Na+ gradient on contractility in skeletal muscle: role of the Na+-K+ pump.
Pflügers Arch.
434:
457-465,
1997[Medline].
33.
Parker, J. C.,
and
J. F. Hoffman.
The role of membrane phosphoglycerate kinase in the control of glycolytic rate by active cation transport in human red blood cells.
J. Gen. Physiol.
50:
893-916,
1967
34.
Paul, R. J.,
M. Bauer,
and
W. Pease.
Vascular smooth muscle: aerobic glycolysis linked to sodium and potassium transport processes.
Science
206:
1414-1416,
1979[Medline].
35.
Paul, R. J.,
C. D. Hardin,
L. Raeymaekers,
F. Wuytack,
and
R. Casteels.
Preferential support of Ca2+ uptake in smooth muscle plasma membrane vesicles by an endogenous glycolytic cascade.
FASEB J.
3:
2298-2301,
1989
36.
Pellerin, L.,
and
P. J. Magistretti.
Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization.
Proc. Natl. Acad. Sci. USA
91:
10625-10629,
1994
37.
Pfliegler, G.,
I. Szabo,
and
T. Kovacs.
The influence of catecholamines on Na,K transport in slow- and fast-twitch muscles of the rat.
Pflügers Arch.
398:
236-240,
1983[Medline].
38.
Richter, E. A.,
N. B. Ruderman,
and
H. Galbo.
Alpha and beta adrenergic effects on metabolism in contracting, perfused muscle.
Acta Physiol. Scand.
116:
215-222,
1982[Medline].
39.
Scholnick, P.,
D. Lang,
and
E. Racker.
Regulatory mechanisms in carbohydrate metabolism.
J. Biol. Chem.
248:
5175-5182,
1973
40.
Schrier, S. L.
Organization of enzymes in human erythrocyte membranes.
Am. J. Physiol.
210:
139-145,
1966[Medline].
41.
Schulz, A. R.
Control analysis of muscle glycogen metabolism.
Arch. Biochem. Biophys.
353:
172-180,
1998[Medline].
42.
Tashiro, N.
Effects of isoprenaline on contraction of directly stimulated fast and slow skeletal muscle of the guinea-pig.
Br. J. Pharmacol.
48:
121-131,
1973[Medline].
43.
Wardlaw, G. M.
The effect of ouabain on basal and thyroid hormone-stimulated muscle oxygen consumption.
Int. J. Biochem.
18:
279-281,
1986[Medline].
44.
Weiss, J.,
and
B. Hiltbrand.
Functional compartmentation of glycolytic versus oxidative metabolism in isolated rabbit heart.
J. Clin. Invest.
75:
436-447,
1985[Medline].
45.
Whittam, R.,
and
M. E. Ager.
The connexion between active cation transport and metabolism in erythrocytes.
Biochem. J.
97:
214-227,
1965.
46.
Whittam, R.,
M. E. Ager,
and
J. S. Wiley.
Control of lactate production by membrane adenosine triphosphatase activity in human erythrocytes.
Nature
202:
1111-1112,
1964.
47.
Wiseman, R. W.,
T. S. Moerland,
P. B. Chase,
R. Stuppard,
and
M. J. Kushmerick.
High-performance liquid chromatographic assays for free and phosphorylated derivatives of the creatine analogues beta-guanidopropionic acid and 1-carboxy-methyl-2-iminoimidazolidine (cyclocreatine).
Anal. Biochem.
204:
383-389,
1992[Medline].
48.
Xu, K. Y.,
and
L. C. Becker.
Ultrastructural localization of glycolytic enzymes on sarcoplasmic reticulum vesticles.
J. Histochem. Cytochem.
46:
419-427,
1998
49.
Xu, K. Y.,
J. L. Zweier,
and
L. C. Becker.
Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport.
Circ. Res.
77:
88-97,
1995
50.
Young, A.,
R. Pittner,
B. Gedulin,
W. Vine,
and
T. Rink.
Amylin regulation of carbohydrate metabolism.
Biochem. Soc. Trans.
23:
325-331,
1995[Medline].
51.
Young, A. A.,
G. J. S. Cooper,
P. Carlo,
T. J. Rink,
and
M. W. Wang.
Response to intravenous injections of amylin and glucagon in fasted, fed, and hypoglycemic rats.
Am. J. Physiol.
264 (Endocrinol. Metab. 27):
E943-E950,
1993
52.
Young, A. A.,
D. M. Mott,
K. Stone,
and
G. J. Cooper.
Amylin activates glycogen phosphorylase in the isolated soleus muscle of the rat.
FEBS Lett.
281:
149-151,
1991[Medline].