Stimulation of both aerobic glycolysis and Na+-K+-ATPase activity in skeletal muscle by epinephrine or amylin

J. Howard James1, Kenneth R. Wagner2, Jy-Kung King1, Rebecca E. Leffler1, Radha Krishna Upputuri4, Ambikaipakan Balasubramaniam1, Lou Ann Friend1, Daniel A. Shelly3, Richard J. Paul3, and Josef E. Fischer1

1 Departments of Surgery, 2 Neurology, and 3 Molecular and Cellular Physiology, University of Cincinnati, Cincinnati 45267; Medical Research Service, Department of Veterans Affairs Medical Center, Cincinnati 45220; and 4 Shriners Hospital for Children, Cincinnati, Ohio 45229


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 × 10-7 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.

ouabain; lactate; oxygen consumption; metabolic compartmentation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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METHODS
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Muscle Incubations

Male Sprague-Dawley rats, weighing 45-55 g, were obtained from Harlan Laboratories (Indianapolis, IN) and used when they weighed between 55 and 60 g (4 wk of age), at which time the extensor digitorum longus (EDL) and soleus muscles weigh between 20 and 25 mg. Rats were fed Mouse/Rat Diet 7012 (Harlan Teklad, Madison, WI) and were provided tap water ad libitum. Lighting was on a 12:12-h light-dark cycle. Hindlimb muscles were dissected with intact tendons from rats anesthetized with pentobarbital sodium (50 mg/kg). Muscles were gently blotted and weighed immediately after dissection. Experimental conditions were as previously described (17), with the modification that muscles were incubated in stoppered 25-ml flasks (22). Muscles were incubated in Krebs-Henseleit buffer containing (in mM) 118 NaCl, 4.6 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, and 10 D-glucose, in a 95% O2-5% CO2 atmosphere. Previous studies showed that a PO2 of >400 mmHg is maintained in the medium over a 2-h incubation in these flasks (22). Muscles were incubated in a shaking water bath at 30°C, rather than 37°C, to slow the metabolic rate and to ensure adequate O2 delivery by diffusion into the muscles (2, 17). To reduce contamination by blood and to equilibrate extracellular fluid with the medium, muscles were preincubated (~3 ml) for 30 min before transfer to fresh medium (3.0 ml) for a 1-h incubation. In studies in which muscle levels of ATP, creatine phosphate, and glycogen were determined, the incubation period was lengthened to 2 h. Ouabain (1 mM), when present, was added to both the preincubation and incubation flasks to allow adequate time for diffusion into extracellular spaces and for binding to the Na+-K+ pumps. Epinephrine and amylin were not present in preincubation but were added only to the incubation medium. In studies in which glucose was omitted, it was absent from both preincubation and incubation medium.

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 × 10-6 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 (volume approx 1.25 ml) fitted with a polarographic O2 electrode (Instech Laboratories, 125/05) and a magnetic stirrer. Chamber temperature was maintained at 30°C. Muscles were mounted by one tendon to a fixed stainless steel post. The other tendon was fixed to the lever of an isometric force transducer via a thin stainless steel wire and adjusted to a passive tension of 0.5 mN. Muscles were kept in sterile Krebs-Henseleit buffer. Chamber O2 pressure (PO2) was maintained at >500 Torr, well above the calculated critical PO2 (~350-450 Torr) at which the diffusive flux of O2 becomes rate limiting for oxidative phosphorylation (12).

Resting 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 × 10-6 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, -180°C) and to which was then added a 6.35-mm (0.25-in.) stainless steel ball bearing, also precooled to liquid N2 temperature. The tube was then shaken rapidly (~15 s) using a dental amalgamator to pulverize the tissue-methanol mixture. The tube was recooled in liquid N2, and shaking was repeated. To the powdered sample was added 1.0 ml of 0.02 N HCl (in two 0.5-ml aliquots) to facilitate sample transfer. A 0.2-ml portion was removed and heated to 100°C for 10 min for later glycogen analysis by use of amyloglucosidase digestion (27). To the rest of the sample 112.5 µl of 3.0 N HClO4 were added, and the sample was centrifuged. Then, 225 µl of 2.5 M KHCO3 were added to each sample to precipitate perchlorate ions, and the sample was centrifuged again. The resulting supernate was filtered, and a 50-µl portion was analyzed by HPLC.

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-(1---37) was synthesized using a solid-phase technique and purified as previously described (1). Standards for HPLC analysis of ATP and creatine phosphate were from Boehringer Mannheim (Indianapolis, IN). All other chemicals were obtained from Sigma Chemical.

Statistical 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
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

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 × 10-9 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).

                              
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Table 1.   Effect of epinephrine on lactate production, [Na+]i, and Na+/K+ in EDL and soleus muscles in absence or presence of ouabain

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 (10-9 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).

                              
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Table 2.   Effect of amylin on lactate production, [Na+]i, and Na+/K+, in EDL and soleus muscles in absence or presence of ouabain

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 · g-1 · h-1, means ± SE, P < 0.0001] and soleus [no ouabain (n = 71) vs. ouabain (n = 56): 3.4 ± 0.1 vs. 2.4 ± 0.1 µmol · g-1 · h-1, means ± SE, P < 0.0001]. Thus the ouabain-inhibitable basal lactate production of EDL was about twice that of soleus. [Na+]i and Na+/K+ were significantly higher in the presence of ouabain than in its absence in both EDL and soleus (Tables 1 and 2). When only the control muscles are considered, ouabain caused a significant decrease in [K+]i [no ouabain vs. ouabain: (EDL) 79.7 ± 1.2 vs. 47.4 ± 1.4 µmol/g, means ± SE, (n = 61 vs. 42), P < 0.0001; (soleus) 70.3 ± 1.3 vs. 37.7 ± 0.9 µmol/g, means ± SE, (n = 61 vs. 42), P < 0.0001].

Epinephrine. 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%.


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Fig. 1.   Epinephrine-stimulated lactate production in extensor digitorum longus (EDL, A) and soleus (B) in the absence (-) or presence (+) of ouabain. Lactate production due to epinephrine was calculated by subtracting lactate production of untreated (Control) muscle from that of contralateral muscle exposed (Exp) to epinephrine. Data are means ± SE for no. of muscles indicated in Table 1. * Differs significantly from epinephrine-stimulated lactate production of ouabain-treated muscle exposed to the same epinephrine concentration, P <=  0.05. dagger  Differs significantly from lactate production by muscles in same ouabain-treatment group in the presence of lowest concentration (5 × 10-9 M) of epinephrine, P <=  0.05.

In the EDL muscles incubated with ouabain, no significant effect of epinephrine on [Na+]i or Na+/K+ could be detected, indicating effective inhibition of the Na+-K+-ATPase. In contrast, soleus muscles treated with epinephrine (5 × 10-8, 5 × 10-7, and 5 × 10-6 M) in the presence of ouabain had significantly lower [Na+]i than their paired control muscles; however, this effect of epinephrine was not reflected in a statistically significant decrease in Na+/K+ (Table 1).

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 · g-1 · 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 × 10-6 M) or ouabain, samples of incubation medium were withdrawn at 15-min intervals, and cumulative lactate production was calculated (Fig. 2). Under the various incubation conditions for both EDL and soleus, lactate accumulated in the medium in an approximately linear manner during the 1-h incubation. For both EDL and soleus, in either the absence or the presence of ouabain, epinephrine significantly increased cumulative lactate production at each of the four 15-min periods (P < 0.02, at least). The statistical significance of the effect of ouabain on lactate production, in either the absence or the presence of epinephrine, is indicated in Fig. 2. These results indicate that the effect of epinephrine on lactate production was apparent within 15 min and continued throughout the entire 1-h period.


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Fig. 2.   Cumulative lactate production, assessed at 15-min intervals, in EDL (A and B) and soleus (C and D), in absence of epinephrine (A and C) and in its presence (5 × 10-7 M; B and D) and in absence (open bars) or presence (shaded bars) of ouabain (Ouab). Data are means ± SE for 6-7 muscles/group. * Differs significantly from lactate production of muscles exposed to ouabain in same time interval and in same panel, P <=  0.05.

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).


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Fig. 3.   Relationship of lactate production to ratio of Na+ to K+ (Na+/K+) in EDL (A) and in soleus (B). Main figure in each panel represents individual values from epinephrine- or amylin-treated muscles; inset represents those from control, untreated muscles. In both main and inset figures, data from muscles treated with ouabain are represented by open circle , without regard for whether those muscles were simultaneously exposed to epinephrine or ouabain. In main figures, muscles treated with epinephrine are represented by ; those exposed to amylin are represented by shaded circles. In insets (Control), symbols represent treatment of muscle to which control muscle was paired. Straight lines represent least-squares best fit of lactate production to Na+/K+, with consideration of epinephrine- and amylin-treated muscles together. It can be seen that the range of values of Na+/K+ was smaller in EDL than in soleus and, therefore, that the slope of the regression line relating lactate production to Na+/K+ was steeper for EDL than for soleus.

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 × 10-6 M) for 30 min during incubation in K+-free medium. Because K+ can be released from the muscles into the medium during incubation, inhibition of the Na+-K+-ATPase by incubation in K+-free medium is unlikely to be as effective as inhibition by 10-3 M ouabain. Nonetheless, in K+-free medium, the Na+/K+ of both EDL and soleus was significantly higher than in medium with normal K+ concentration, indicating inhibition of the Na+-K+-ATPase (Fig. 4). The stimulation of lactate production by epinephrine was also significantly reduced in K+-free medium by 30-40% (Fig. 4).


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Fig. 4.   Effect of K+-free medium on lactate production (left) and intracellular Na+/K+ (right) in muscles incubated with epinephrine (5 × 10-6 M). Data are means ± SE for 7-10 muscles/group. * Differs significantly from value in same muscle incubated in normal medium, P <=  0.05.

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 × 10-6 M). Rates of O2 consumption obtained in the present studies were consistent with values previously obtained by others using similar procedures in soleus muscle from rats of the same age (43). Addition of epinephrine had no significant effect on O2 consumption (Table 3); lactate production by the muscles in which O2 consumption was measured was significantly increased after epinephrine addition (data not shown).

                              
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Table 3.   Effect of epinephrine (5 × 10-6 M ) on lactate production, tissue content of glycogen, creatine phosphate, ATP, and O2 consumption of EDL and soleus muscles in absence or presence of ouabain

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 × 10-6 M) (Table 3). Creatine phosphate and ATP levels in all cases were consistent with previously reported values for resting rat EDL and soleus muscles, indicating that muscle energy production and viability were well maintained during the incubations. After exposure to epinephrine in the absence of ouabain, glycogen content was significantly reduced by ~50% in EDL and by ~43% in soleus (Table 3). In the presence of ouabain, epinephrine resulted in only a 29% reduction of glycogen in EDL and only a 12% reduction of glycogen in soleus. Glycogen was significantly higher in muscles exposed to both epinephrine and ouabain compared with those treated with epinephrine alone (Table 3).

Differences 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-1 · h-1, means ± SE, P < 0.0001]. In these untreated, control muscles, the [Na+]i of EDL was significantly lower than that of soleus [EDL (n = 61) vs. soleus (n = 61): 5.9 ± 0.1 vs. 11.9 ± 0.2 µmol/g, means ± SE, P < 0.0001]. Similarly, Na+/K+ of EDL muscles (n = 61) was significantly lower than that of soleus (n = 61) (EDL vs. soleus: 0.075 ± 0.002 vs. 0.171 ± 0.003 µmol/g, means ± SE, P < 0.0001). Differences between EDL and soleus in Na+ and K+ content have been noted previously (17, 18); however, a significant difference in basal lactate production between EDL and soleus has not been reported previously.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 beta -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 × 10-6 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 (10-6 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 × 10-6 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 × 10-6 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 · g-1 · 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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