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CELLULAR METABOLISM
1Department of Kinesiology, Arizona State University, Tempe, Arizona; and 2Department of Kinesiology, Kansas State University, Manhattan, Kansas
Submitted 3 April 2007 ; accepted in final form 11 October 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) in the vicinity of 30 s. The mechanisms underlying this delay have been studied over several decades. Meyer's electrical analog model proposed the concept that the is given by = Rm·C, where Rm is mitochondrial resistance to energy transfer, and C is metabolic capacitance, determined primarily by the cellular total creatine pool (TCr = phosphocreatine + creatine). The purpose of this study was to evaluate in vitro the Jo kinetics of isolated rat skeletal muscle mitochondria at various levels of TCr and mitochondrial protein. Mitochondria were incubated in a medium containing 5.0 mM ATP, TCr pools of 0–1.5 mM, excess creatine kinase, and an ATP-splitting system of glucose + hexokinase (HK). Pyruvate and malate (1 mM each) were present as oxidative substrates. Jo was measured across time after HK was added to elicit one of two levels of Jo (40 and 60% of state 3). At TCr levels (in mM) of 0.1, 0.2, 0.3, 0.75, and 1.5, the corresponding values (s, means ± SE) were 22.2 ± 3.0, 36.3 ± 2.2, 65.7 ± 4.3, 168.1 ± 22.2, and 287.3 ± 25.9. Thus increased linearly with TCr (R2 = 0.916). Furthermore, the experimentally observed varied linearly and inversely with the mitochondrial protein added. These in vitro results consistently conform to the predictions of Meyer's electrical analog model. mitochondrial resistance; hexokinase; creatine kinase
In 1988, Meyer (46) proposed an electric analog model of oxidative phosphorylation in which the total creatine pool [TCr = phosphocreatine (PCr) + creatine (Cr)] in muscle acts as a metabolic capacitor. According to this model, the time constant () of Jo kinetics depends on the mitochondrial resistance to energy transfer (Rm) and the magnitude of the TCr pool i.e., the metabolic capacitance (C):
 | (1) |
GATP) and mitochondrial Jo (9, 11, 34, 43), together dictate that a predictable PCr net breakdown (a discharge of capacitance) must occur in the transition from one (lower) steady-state ATP turnover to another (higher) rate. That negligible C exists within the mitochondrion itself, e.g., in the matrix, electron transport chain, or proton gradient, has been shown by Wojtczak et al. (66, 67). Thus the strictest interpretation of the Meyer model predicts that any impact mitochondria make on the kinetics of O2 uptake would be evident in the steady-state Jo-GATP relation. In contrast, TCr would be predicted to affect neither the energetic forces nor flows once steady state is achieved. Meyer experimentally supported his model by showing that progressive depletion of the Cr pool of rat skeletal muscle with dietary β-guanidinopropionic acid (β-GPA) yields faster PCr kinetics, as assessed with 31P-nuclear magnetic resonance spectroscopy, at the onset of elevated contractile demand (44). More recently, the model has been supported in computer models of oxidative phosphorylation (35). In addition, Kindig et al. (31) reported much faster Jo kinetics in isolated myocytes in the presence of a CK inhibitor. It is important to note, however, that elevating cellular TCr, via dietary Cr supplementation, failed to slow Jo kinetics in exercising human muscle (29), as the Meyer model would predict.
A different conceptual approach to what accounts for the sluggish cellular adjustment to ATP demand describes the response as "metabolic inertia" (18, 22, 26, 50). In this view, a lag in the availability of oxidative substrate accounts for the delay in the matching of oxidative phosphorylation to ATP demand (22, 26, 50, 55). It is difficult to envision how a dynamically changing fuel availability could independently give rise to monoexponential Jo kinetics. On the other hand, to the extent that fuel availability modifies the fall in GATP required to achieve steady state, it would correspondingly alter Jo kinetics via the coupling between PCr discharge and GATP. Accordingly, we have recently shown (43), as Koretsky and Balaban (34) showed earlier, that fuel availability does indeed influence the steady-state GATP-Jo relation. Viewed in this way, metabolic inertia essentially reduces to Meyer's more general model.
The kinetics of PCr breakdown and Jo rise are virtually identical at the onset of exercise (1, 2, 35, 39, 42, 51, 62) because of a relatively constant phosporylation ratio during myocyte contraction. Therefore, the purpose of this study was to evaluate Jo kinetics using a novel in vitro system in which isolated skeletal muscle mitochondria were exposed to an ATP-splitting system [glucose + hexokinase (HK)] and varying amounts of TCr. In separate incubations, varying amounts of TCr and mitochondrial protein were added to a respiration chamber to experimentally manipulate C and resistance to energy transfer, respectively. This experimental approach provides the tools to extend the observations of previous reports because it allows for the direct manipulation of the ratio of TCr mass to mitochondrial content to values both below physiological, as previously shown in the β-GPA and CK inhibitor studies, and well above physiological, which has not been previously tested (for quantitative analysis, see DISCUSSION). Moreover, the model enables complete and instantaneous control of all oxidative substrates, a feature that can be used to explore the metabolic inertia hypothesis.
We hypothesized that mitochondria interacting with a TCr pool would conform to the behavior of an RC electrical circuit. We assessed the validity of this hypothesis by testing the predictions that logically arise from it: 1) of the Jo response to a step increase in ATP breakdown rate would vary linearly with the TCr in the system, 2) would vary linearly with the Rm to energy transfer in the system, and 3) would be independent of the ATP turnover rate.
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MATERIALS AND METHODS |
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Isolation of mitochondria. Mitochondrial isolations were performed at 0–4°C according to the methods of Makinen and Lee (40). Excised muscles were trimmed to remove fat and connective tissues and were then minced, weighed, and placed in 9 vol of solution I. Protease (Nagarse; Sigma Chemical, St. Louis, MO) was added (5 mg/g wet muscle), and the digested mince was mixed continually for 7 min. Digestion was terminated through the addition of an equal volume of solution I, and the mince was homogenized with an Ultra-Turrax (Cincinnati, OH) blender for 15 s at 40% of full power. The homogenate was centrifuged at 700 g for 10 min in a refrigerated centrifuge (model J2–21M/E; Beckman) to pellet down contractile protein and cellular debris. The supernatant was rapidly decanted through a double layer of cheesecloth and centrifuged at 14,000 g for 10 min to pellet down the mitochondrial fraction. The supernatant was discarded; the mitochondrial pellet was resuspended and washed in a volume equal to the original homogenate in a solution containing (in mM) 100 KCl, 40 Tris·HCl, 10 Tris base, 1 MgSO4, 0.1 EDTA, 0.2 ATP, and 2% (wt/vol) BSA (no. A-7030, fatty acid content <0.01%; Sigma Chemical), pH 7.40 (solution II); and the suspension was centrifuged at 7,000 g for 10 min. The supernatant was discarded, and the pellet was resuspended in 20 ml of a solution similar to solution II, but without BSA (solution III). This resuspended pellet was subsequently centrifuged at 3,500 g for 10 min.
The final mitochondrial pellet was suspended in 400–500 µl of a solution containing (in mM) 220 mannitol, 70 sucrose, 10 Tris·HCl, and 1 EGTA, pH 7.40, yielding a final protein content of 5.0–11.5 mg mitochondrial protein/ml (38).
Mitochondrial respiration. Mitochondrial Jo was measured as described by Messer et al. (43). Briefly, Jo was measured polarographically in a respiration chamber maintained at 37°C (Rank Brothers, Cambridge, UK). Incubations were carried out in a 2.0-ml final volume of respiration medium (RM) adapted from Wanders et al. (59). The basic RM contained (in mM) 100 KCl, 50 MOPS, 20 glucose, 10 K2PO4, 10 MgCl2, 1 EGTA, and 0.2% BSA, pH 7.00.
Functional integrity of isolated mitochondria. Mitochondria, 100–200 µg protein, were added to 2.0 ml RM with 1 mM pyruvate plus 1 mM malate as oxidative substrates. State 3 (maximal) Jo was initiated with a bolus addition of 1 µmol ADP. State 3 and state 4 (resting) Jo, as described by Estabrook (15), were measured, and the respiratory control ratio (RCR) was calculated as the ratio of state 3-to-state 4 respiration. The ADP-to-oxygen ratio was also determined (15).
HK-stimulated submaximal Jo.
The kinetic experiments required the establishment of steady-state ATP breakdown rates corresponding to known fractions of the mitochondrial aerobic capacity. Thus, after state 4 respiration ensued in the mitochondrial integrity incubation described above, the relationship of added HK to elicited Jo was determined. First, ATP was added to give a 5 mM final concentration. Next, stepwise additions of yeast HK were made while Jo was followed continuously to determine the HK-Jo relationship. HK-driven oxidative phosphorylation is briefly summarized as (stoichiometry omitted):
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 | (3) |
Thus, a steady-state Jo can be experimentally established with the appropriate HK addition.
Kinetic experiments.
Kinetic studies examined the influence of variable capacitance (TCr added) and resistance (mitochondrial protein added) on the approach of Jo to steady state after a step increase in ATP turnover elicited by HK addition. In these kinetic experiments, it was critical for all mitochondrial preparations to begin in the same state of resting Jo and energetic driving forces. To this end, each preparation was preincubated for at least 10 min in the presence of oxidative substrates and the appropriate pools of adenylate and Cr energy phosphates. Accordingly, for the capacitance studies, in 5.0 ml of basic RM, the following additions were made (in mM): 1.0 pyruvate, 1.0 malate, and 5.0 ATP. TCr, at a ratio of PCr/Cr = 2, was then added at one of six levels (0, 0.1, 0.2, 0.3, 0.75, or 1.5 mM) and finally 75 U CK/ml. Mitochondria, 50–100 µg/ml, were then allowed to equilibrate for 10 min. This preincubation ensured that, before HK addition, near-thermodynamic equilibrium was obtained between mitochondrial driving forces and the energy phosphate pools linked through the CK reaction:
 | (4) |
60% state 3 Jo) or a low (40% state 3 Jo) respiration rate. Jo was then followed continuously for 6 min. For the Rm studies, the capacitance procedures described above in Kinetic experiments were basically followed but were modified by using a range of mitochondrial content (50–350 µg/ml), only two levels of TCr (0.15 and 0.3 mM), and one standard HK addition, which elicited the same steady-state absolute Jo in all incubations (32.84 ± 4.94 nmol O2/min).
Metabolite concentrations. After the 10-min incubation (resting) and exactly 6 min after the HK addition (submaximal steady-state Jo), 500 µl of the experimental medium were aspirated, immediately added to 150 µl of 17.5% HClO4, and centrifuged at 14,000 g for 1 min. The acidified supernatant (500 µl) was quickly added with vigorous mixing to 125 µl of 2.0 N KOH and 0.30 M MOPS. This neutralized extract was stored at –80°C for subsequent determination of metabolite concentrations.
ATP was assayed using enzyme reactions linked to the oxidation or reduction of NADP+/NADPH as described previously by Bergmeyer (8). PCr and Cr were assayed colorimetrically (13).
Free ADP (ADPf) was calculated using the ATP, PCr, and Cr concentrations and a CK equilibrium constant (KCK) of 150 (17).
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The cytosolic GATP was calculated using the ATP and ADP concentrations and the experimentally added Pi concentration of 10 mM
 | (6) |
is the standard free energy of ATP hydrolysis (–7,592 cal/mol), R is the gas constant (1.987 cal·deg–1·mol–1), T is degrees Kelvin (17), and brackets denote concentration.
Kinetic analysis.
Jo kinetics were analyzed using the model by Barstow et al. (1)
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is in seconds. It should be noted that our experimental model permitted the unequivocal determination of JHK(ss) from the daily HK-Jo relationship we determined, as described under HK-stimulated submaximal Jo. Using the known JHK(ss) and the measured Jo(t), Jo(rest), and time values, was calculated using Eq. 5 and nonlinear regression with SPSS statistical software (version 13.0; SPSS, Chicago, IL).
For the capacitance studies, the mitochondrial content added to the system varied slightly from day to day (6 experiments/daily preparation) because of differences in the final mitochondrial protein concentration across days. Differences in mitochondrial content would be predicted to alter the Rm to energy transfer; this assumption was experimentally verified (see Influence of mitochondrial content on Jo kinetics). Accordingly, values were normalized to the average daily mitochondrial content by multiplying the measured value for a given run by the ratio of mitochondrial content of that run to the average mitochondrial content (0.15 mg).
Statistical analysis. Significant differences were identified using two-way ANOVA (Jo vs. TCr). A Levene test of equality of error variances was used to determine if the variances could be assumed to be equal. When the variances could be assumed to be equal, a Tukey honestly significant difference post hoc test was used to test for differences between specific mean values. If the variances could not be assumed equal, a Games-Howell post hoc test was used. P values <0.05 were taken as statistically different.
A least-squares regression line was determined for the -TCr relationship and the -Rm to energy transfer relationship using SPSS.
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RESULTS |
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Rest. In the presence of a 5 mM adenylate pool, resting Jo was 115.1 ± 14.6 O2·mg–1·min–1, or 19.2 ± 1.6%, of state 3 Jo. Resting Jo was not influenced by the TCr or the mitochondrial content in the medium.
Before HK addition, GATP, [ATP], and the [ADPf] were not different across all TCr conditions (Table 1). The sum of the assayed [Cr] and [PCr] reflected the experimental addition of TCr, but the PCr-to-Cr ratio established by the resting mitochondria during the preincubation was greater than the PCr/Cr of 2.0 that was added. At 0.1, 0.2, 0.3, 0.75, and 1.5 mM TCr the PCr/Cr established by mitochondrial driving forces during the preincubation were 4.55 ± 0.63, 4.55 ± 0.27, 5.66 ± 0.49, 6.50 ± 0.94, and 5.44 ± 0.53, all greater than 2.0 and none of which were different from each other. Mitochondrial content had no effect on resting energy phosphate concentrations (Table 2).
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The Jo elevation above rest elicited by HK addition was achieved at the expense of higher ADPf and lower GATP. Figure 1 shows the characteristic GATP-Jo relationship (9, 11, 34, 43) and further shows that the TCr of the incubation medium did not influence the force-flow characteristics. In fact, neither [ADPf] nor GATP for a given Jo was influenced by TCr. [Cr] increased in proportion to Jo for all levels of TCr, whereas [PCr] decreased. Neither the TCr added nor the HK-stimulated Jo had any affect on [ATP]. As explained above in Steady state, steady-state energy phosphates at 0.75 and 1.5 mM TCr are not reported because steady-state Jo was not attained.
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GATP and smaller increases in [ADPf] in transition to steady-state Jo (Table 2). These smaller changes in energy status also resulted in roughly one-half the increases in [Cr] and half the decreases in [PCr].
Kinetic analysis of Jo.
The rose systematically with increasing TCr (Table 3). Furthermore, at a given TCr, was not influenced by ATP turnover rate (low vs. high HK addition). When all data are regressed against TCr, the relation between and TCr was linear (R2 = 0.915), as shown in Fig. 2. Furthermore, the mean R2 for each individual preparation was 0.921. In addition, the relationship between and Rm (Fig. 3) was also linear (R2 = 0.999 and 0.941 for 0.15 and 0.30 mM TCr, respectively), with the slope varying according to the added TCr (8.81 and 16.04 mg·s for 0.15 and 0.30 mM TCr, respectively).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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of the Jo response to HK addition varied linearly with the TCr present in the incubation. Moreover, was also linearly, and inversely, related to the mass of mitochondrial protein added to the incubation. Thus, under the experimental conditions used in these studies, the kinetics of mitochondrial Jo conformed to the electrical analog model proposed by Meyer (46).
Influence of TCr on resting mitochondria.
At rest, the TCr mass in the medium influenced neither the energetic force (GATP) maintained by the mitochondria nor the metabolic flux (Jo). Thus the mitochondria were in the same energetic condition before HK addition regardless of the TCr condition. During the 
10-min preincubation period, all mitochondria carried out a net phosphorylation of added Cr such that the experimentally added PCr-to-Cr ratio of 2.0 was observed to rise to a steady-state resting mean ratio of 5.3 across all conditions (see Table 1). These in vitro PCr-to-Cr ratios are slightly higher than the resting in vivo values of type II muscle reported by Kushmerick et al. (37), which might be attributable to the saturating oxidative substrate levels used in our in vitro model (43). High structural and functional integrity of the isolated mitochondria used in these studies is certainly implied by the robust energetic state supported by them.
Influence of TCr on steady state mitochondrial Jo. Similar to the resting condition, at both levels of HK addition, the energy phosphate concentrations and the steady-state Jo were not different across TCr levels (Fig. 1). Combined, these data suggest that TCr does not influence respiration in the steady state. According to the chemiosmotic theory of oxidative phosphorylation, respiration rate depends on the difference between the mitochondrial redox driving force and the extramitochondrial free energy of ATP hydrolysis, along with the Rm to energy transfer (47). Apparently, in these in vitro preparations, the TCr in the medium had no significant effect on either the net driving force or the apparent conductance of the pathway.
Influence of TCr on Jo kinetics.
The delay in the attainment of steady-state Jo was linearly related to the TCr present in the incubation. The greater the TCr mass in the system, the greater was the hydrolysis of PCr necessary to achieve steady state and, thus, the larger was the of the Jo response to a step increase in ATP demand. In the context of the model, additional TCr in the system corresponds to a larger capacitance that must be discharged during the transition to steady-state Jo. Significantly, the relationship was observed across all TCr conditions examined, spanning sub- to supraphysiological ratios of TCr/mitochondrial content. The results therefore imply that the study by Jones et al. (29), which reported no effect of dietary Cr supplementation on oxygen uptake kinetics in humans, may have been limited methodologically. First of all, muscle TCr was not actually measured in that study; the authors stated that the supplementation regimen used would be expected to result in a 15–20% increase in TCr in the muscle. Second, they did report a numerical, though nonsignificant, 10% increase in , from 17.3 ± 1.7 to 19.0 ± 1.3 s, which may well be more of a confirmation than a challenge to the concept that increased TCr predicts higher .
Influence of mitochondrial content on Jo kinetics.
The transition time to steady-state Jo was also inversely related to the mitochondrial content added to the system. At a given steady-state ATP turnover rate, doubling the mitochondria halved the fall in GATP required to achieve steady state (from 0.526 to 0.292 kcal/mol; see Table 2). Doubling the mitochondria resulted in [ADP] and [Cr] rising one-half as much and [PCr] falling to only one-half the extent (one-half as much PCr was discharged in the transition to steady state). In the context of the model, doubling the mitochondria reduced Rm by a factor of two and thus halved .
If mitochondrial content is inversely proportional to resistance to energy transfer (Rm) and the TCr in the system accounts for essentially all C, then a plot of the measured against the reciprocal of the added mitochondria (mg–1) should yield a slope proportional to C. To test this prediction, we evaluated at two levels of TCr, 0.15 and 0.30 mM, across several levels of mitochondria additions. As can be seen in Fig. 3, increased linearly with the reciprocal of added mitochondrial protein, and doubling the TCr increased the slope of the -Rm regression line 1.8-fold, in good agreement with these predictions.
Conformation to the electric analog model.
One major requirement for the electric analog model to be successful is that steady-state [PCr] must be linearly related with the steady-state GATP, and, as such, the slope of this relationship is equal to C. This can then be further simplified to show that C = TCr/6RT (46). For example, using the data from the double mitochondria trial in Table 2, the capacitance is 1.6 x 10–7 mol2/kcal using either of the above methods. A second major requirement for the model is that the steady-state rate of oxidative phosphorylation (Jp) is linearly related to the steady-state GATP. If we assume a constant ATP/O2 (i.e., that the measured Jo remains proportional to Jp across all GATP), then this linearity was experimentally observed, as shown in Fig. 1. The reciprocal of the slope of the force-flow relationship can then be used to calculate Rm by using the chemiosmotic theory of oxidative phosphorylation. Once again, using data from Table 2, when flow (Jo) is expressed in absolute units, and an ATP-to-O2 ratio of 5.0 is assumed, Rm is 4.9 x 106 and 2.9 x 106 kcal·min·mol–2 for single and double mitochondria trials, respectively. Together, these data show that adding more mitochondrial protein to the incubation reduced Rm and in proportion, in accordance with Eq. 1.
The steady-state data of Table 2 can be further used to show that indeed equals the product of Rm and C, at least under these in vitro conditions. For example, in the double mitochondria trial, Rm (2.9 x 106 kcal·min·mol–2) multiplied by C (1.6 x 10–7 mol2/kcal) gives a value of 0.46 min, or 27.8 s. The experimentally measured value was nearly identical to this prediction, 28.4 s. In addition, if Rm is dGATP/dJp and C is d[PCr]/dGATP, then Rm·C would be equal to d[PCr]/dJp. Thus, knowing simply the change in [PCr] and the change in Jo and assuming a constant ATP-to-O2 ratio, a quick approximation of the can be made. Using the data from the double mitochondria trial gives a calculated of 30.0 s. Furthermore, the relationship between the fall in [PCr] and the rise in Jo can explain why the is the same for both the high and low ATP turnover conditions. Although a greater initial "acceleration" of Jo (30) would be expected with a higher ATP turnover rate (a faster accumulation of ADP), the relationship between the changes in [PCr] and Jo, i.e., the sensitivity of respiratory control, should remain the same. Indeed, in the presence of 0.1 mM TCr, [PCr] fell by 13 and 25 µM after the low and high HK additions, respectively (Table 1), whereas Jo increased by 129.1 and 259.6 nmol·mg–1·min–1 after the low and high HK additions, respectively. The changes in [PCr] and Jo each matched the given stimulus, whereas the d[PCr]/dJo ratio remained the same, thus explaining the similar between the two ATP turnover conditions (Table 3).
As noted above, in calculating Rm, an ATP-to-O2 ratio of 5.0 was assumed, and, consequently, the Rm values are dependent upon this assumption. In contrast, the values for C (1.6 x 10–7 mol2/kcal) calculated above were independent of the ATP-to-O2 ratio. Notably, if C is calculated as /Rm again using Table 2 data, the value for C becomes dependent on the ATP-to-O2 ratio as well, yet nevertheless yields the same value, C = 1.6 x 10–7 mol2/kcal. Thus the same value for C is attained whether the calculation is dependent or independent of the ATP-to-O2 ratio, providing compelling evidence that the ATP-to-O2 ratio was indeed 5.0.
Influence of ATP turnover rate on Jo kinetics.
In vivo, oxygen uptake during high-intensity exercise fails to conform to monoexponential kinetics, instead following a double exponential model where a slow component is observed, elevating and delaying the attainment of steady-state Jo (4, 60). It has been proposed that the slow component results from the simultaneous recruitment of types I and II muscle fibers at the onset of exercise. Type I fibers achieve steady state rapidly, accounting for the fast component of Jo adjustment, and, as exercise duration proceeds, the slow kinetics of type II fibers become apparent (3, 61). In most vertebrates studied, compared with the type I cell, type II myocytes have nearly two times the TCr content (37) and almost three times lower mitochondrial content (27). Thus Jo kinetics in type II muscle would be predicted to be approximately six times slower than in type I muscle (10). In fact, the for the initial fast component of Jo kinetics is reported to be roughly six times faster than that of the slow component (3, 4, 57). It should be mentioned that it has also been proposed that the slow component may be due to sequential, rather than instantaneous, recruitment of type II fibers. In this case, the apparent for a step change in Jo may be due to different motor units being recruited as time goes on, the net effect being a gradually growing slow component (5–7).
Relationship to in vivo conditions. These in vitro experiments utilize mitochondria that have been removed from the structured environment within the cell. Isolation and incubation within an aqueous medium undoubtedly influences both mitochondrial ATP production and the means by which ADP is delivered to the phosphorylating mechanism (48, 54). Nevertheless, with sufficient caution, we believe the findings reveal important features of mitochondrial function. Furthermore, the robust energetic forces and flows maintained by the isolated mitochondria are similar to those observed in vivo (9, 14, 28, 45) and attest to their structural and functional integrity surviving the isolation procedure.
These experiments were designed with the hope of achieving a range of that spanned those observed in intact muscle and whole body systems. Thus TCr (mM) and mitochondrial protein (mg) were added to the respiration chamber at ratios below and above the ratios of capacitance to resistance in intact muscle. Specifically, mixed human skeletal muscle contains 30 µmol TCr/g muscle (41, 58) and 10 mg mitochondrial protein per gram wet muscle (49), giving a ratio of 3.0 (µmol TCr/mg mitochondrial protein). Because we routinely incubate 0.1 mg mitochondrial protein in our assays, a range of TCr above and below 0.3 µmol was employed. The fact that the experimentally observed values were indeed in the physiological range (29, 32, 33) reveals the following two noteworthy findings: 1) the resistance to energy transfer in these isolated mitochondria under in vitro conditions is similar to that in vivo and 2) TCr accounts for essentially all of the capacitance in the transition from one steady-state rate of oxidative phosphorylation to another.
The decision to use an ATP concentration of 5 mM in the in vitro system was aimed to avoid kinetic limitations of low ADP availability (12). Relative to the mitochondrial addition to the incubation chamber (0.1 mg protein), therefore, the adenylate pool was unphysiologically high by a factor of roughly 300. As a consequence, in the absence of added TCr, there was a measurable time lag between HK addition and the attainment of steady-state Jo; averaged 15.1 ± 1.3 s with no TCr in the system. Net hydrolysis of ATP and the response time of the polarography unit could together account for this value. Specifically, the fall in GATP necessary to elicit a given Jo required a net hydrolysis of ATP to ADP that could account for approximately one-half of the delay (7.8 ± 0.6 s). In separate runs, the response time of the polarography unit was evaluated by adding a bolus of NADH to the respiration chamber containing RM and very high activity NADH oxidase (inner membrane prepared by sonicating and concentrating a collection of mitochondrial suspensions). In these experiments, a of 6.6 s was observed as the polarography unit attained a steady-state respiration rate. Taking into account the lag caused by both the ATP capacitance and the response time of the polarography unit, the in the absence of any TCr would be nearly zero (see Table 3). These data, together with the data of Meyer (44) for in situ muscle and Wojtczak et al. (67) in isolated mitochondria, reveal very little capacitance inherent within mitochondria.
As long as the CK reaction is maintained near equilibrium, which at ATP turnover rates within the aerobic scope appears to be the case (36), the duration of the metabolic transient will predictably be substantial, even if all the upstream driving forces for oxidative phosphorylation such as the "redox pressure" and protonmotive force are assumed to remain stable (46). Nevertheless, the term metabolic inertia has recently emerged, proposing that a lag in the availability of oxidative substrate accounts for the delay in the matching of oxidative phosphorylation to ATP demand (22, 26, 50, 55). The primary cause of this version of metabolic inertia is reportedly due to the delayed activation of pyruvate dehydrogenase (PDH) at the onset of exercise. However, the effect of prior activation of PDH is unclear. There are reports that activation of PDH by dichloroacetate (DCA) before exercise elicits the accumulation of acetyl-CoA and spares PCr during the transition from rest to exercise (21, 55, 56). In contrast, it has also been reported that PDH activation by DCA has no effect on the total change in PCr and also results in a slower time course of PCr breakdown (25, 50). There are also conflicting results with respect to the influence of DCA on the for Jo kinetics, since studies done in frog single muscle cells (26), dog gastrocnemius (21), and humans (33) have reported faster, slower, and similar , respectively. A similar approach has shown that PDH activation can be achieved by "priming" exercise before a subsequent moderate exercise bout (23), yet it still remains unclear what the effect is on the for Jo kinetics. Prior exercise has resulted in either a faster (23, 24) or unchanged (16, 53) , yet prior heavy exercise has been reported to necessitate a greater PCr breakdown (23, 53), contrary to the numerous studies showing a coupling between Jo and PCr kinetics (1, 2, 35, 39, 42, 51, 62). Even in those studies in which the for Jo was speeded with prior exercise, this only speeded kinetics by 25% (23) to 36% (24), implying that the majority of the mechanism(s) responsible for the of Jo were unaffected.
Similar to reports by Rasmussen and Rasmussen (48), our isolated mitochondria apparently contain fully dephosphorylated (covalently activated) PDH, because pyruvate-dependent respiration rates across energetic states spanning GATP of –14.5 kcal/mol (approximating in vivo rest condition) to saturating ADP, and across pyruvate concentrations from 10 µM to 1 mM, are not influenced by the addition of 5 mM DCA (43). Thus, although this so-called metabolic inertia could not be expected to operate in our in vitro system in the presence of saturating pyruvate, the experimentally observed did conform to values predicted by the ratio of mitochondrial content and TCr observed in vivo, as described above in Relationship to in vivo conditions. In addition, a modification of the in vitro system used does provide the opportunity to explore the influence of pyruvate-dependent flux through PDH on Jo kinetics. To this end, in additional experiments, we respired mitochondria in the presence of a CK energy clamp (43) to hold the free energy of ATP constant (GATP approximately –14.0 kcal/mol), whereas pyruvate was added stepwise, and the Jo response was recorded continuously (results not shown). Over the entire range of pyruvate concentrations studied (10 µM to a saturating value of 1.0 mM), Jo kinetics consistently proceeded as fast as the polarography unit would allow ( 6–7 s). These results are consistent with the conclusions of Meyer and Wojtczak et al. alluded to above: The flux of PDH products in some potential downstream capacitance, such as the matrix NAD+ or acetyl-CoA pools or the protonmotive force, apparently cannot account for any of the time lag during the metabolic transient.
Summary.
In summary, mitochondria interacting with an added ATP-hydrolyzing system and various levels of added TCr conformed to the behavior of an RC electrical circuit, as predicted by Meyer's model of 1988. We conclude that the of the rise to a steady-state aerobic flux increased linearly with increases in TCr (C), decreased linearly with reduced Rm, and was independent of ATP turnover rate. The first-order Jo kinetics of muscle subjected to a step change in energy demand are largely attributable to, and adequately explained by, the magnitude of the TCr and the Rm to energy transfer.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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