Transgenic livers expressing mitochondrial and cytosolic CK: mitochondrial CK modulates free ADP levels

Nadir Askenasy and Alan P. Koretsky

Department of Biological Sciences, Pittsburgh NMR Center for Biomedical Research, and Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The function of creatine kinase (CK) and its effect on phosphorus metabolites was studied in livers of transgenic mice expressing human ubiquitous mitochondrial CK (CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination. 31P NMR spectroscopy and saturation transfer were recorded in livers of anesthetized mice to measure high-energy phosphates and hepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxes mediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 µmol · g wet wt-1 · min-1), the fraction of phosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrations calculated by assuming equilibrium of the CK-catalyzed reaction in vivo decreased from 84 ± 9 to 38 ± 4 nmol/g wet wt. Calculated free ADP levels in mice expressing high levels of CK-B (920-1,635 µmol · g wet wt-1 · min-1) were 52 ± 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 ± 4 nmol/g wet wt. These findings indicate that CK-Mit catalyzes its reaction equally well in both directions and can lower hepatic apparent free ADP concentrations.

mitochondrial creatine kinase; cytosolic creatine kinase; phosphorus-31 nuclear magnetic resonance spectroscopy; magnetization transfer; liver adenosine 5'-diphosphate concentration


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CREATINE KINASE (CK) catalyzes the transfer of high-energy phosphate between phosphocreatine (PCr) and MgATP. This reaction, PCr + MgADP + H+ left-right-arrow MgATP + creatine (Cr), is catalyzed in both directions by CK. In tissues with abundant CK, such as skeletal muscle, heart, and brain, the rapid reaction maintained by the enzyme (21) enables buffering of ADP, ATP, and pH during periods when ATP utilization exceeds ATP production by glycolysis and oxidative phosphorylation (50). In mammals, five genes are known to encode four isoenzymes of CK. In skeletal muscle the MM dimer of CK is predominant, in brain the BB dimer is predominant, and heart contains the MM dimer and the MB heterodimer. In addition to these cytosolic isoforms of CK, there is a mitochondrial form of CK (CK-Mit) that localizes to the intermembranal space of mitochondria. A sarcomeric form of CK-Mit is expressed in heart and skeletal muscle, and a ubiquitous CK-Mit is expressed in brain and other tissues (50). The specific CK isoenzyme composition of a tissue depends on the developmental and functional characteristics of the tissue. In the fetal stage, CK-BB is almost exclusively expressed, whereas the CK-MM and CK-Mit isoenzymes are features of differentiation (13, 44, 50). The expression of CK-Mit has been correlated with the development of oxidative capacity in heart and brain (9, 49), whereas levels of CK-MM correlate with the glycolytic capacity of muscle (30). The pattern of expression of CK isoenzymes changes during adaptation of muscle, with a decrease in CK-MM and an increase in CK-Mit with processes that convert fast-twitch toward slow-twitch muscle (37). Considering the simple reaction catalyzed by CK, it is not yet clear why there is such a rich diversity in expression of CK isoenzymes.

The most striking difference between CK isoenzymes is their different subcellular localization. CK-Mit localizes to the intermembrane space of mitochondria, where it has been proposed to make a complex with adenine nucleotide translocase (ANT) in the inner membrane and porin in the outer membrane (1, 23, 32, 34). CK-MM and CK-BB are predominantly cytosolic but also show patterns of subcellular localization. CK-MM localizes to the outer face of sarcoplasmic reticulum and to myofibrils, whereas CK-BB has been localized to the plasma membrane and mitotic spindles (10, 31, 35, 38). The pattern of subcellular localization of CK has led to a model, referred to as the PCr circuit or shuttle hypothesis, that postulates coupling of CK-Mit to mitochondrial ATP generation and coupling of CK-MM (or CK-MB or CK-BB depending on the tissue) to ATP utilization (4, 50). The specific localization of CK is thought to buffer against local changes in ATP/ADP levels as well as to affect ATP/ADP delivery, production, and utilization. In particular, there has been a great deal of work studying the role of CK-Mit. Two main sources of experimental evidence support an important role for CK-Mit in coupling alterations in ADP to changes in ATP production by oxidative phosphorylation. First, extensive localization of the enzyme indicates that it is localized at contact sites of the inner and outer mitochondrial membrane in close association with porin and ANT (1, 24, 32, 34, 50). Second, a great deal of biochemical work indicates that ATP made by mitochondria is directly coupled to PCr generation and that the ADP produced is coupled to transport back into the mitochondrial matrix. In isolated mitochondria this coupling results in a lowering of the apparent Km of oxidative phosphorylation for ADP and eliminates the need to transport adenine nucleotides across the outer mitochondrial membrane (49). Evidence indicates that the outer mitochondrial membrane acts as a permeability barrier for ADP (33).

Despite extensive biochemical studies implicating CK-Mit in affecting mitochondrial function, there is little direct in vivo evidence for an important specific role of CK-Mit. Recently both the ubiquitous and sarcomeric forms of CK-Mit have been knocked out in mice. No obvious phenotypes are associated with loss of CK-Mit function (40, 41). There is evidence that elimination of CK causes adaptation of mitochondria, which may mask any direct effects of eliminating CK (49). An alternative strategy is to add CK-Mit to a tissue that does not normally contain it, such as the liver, to determine whether there is a gain of function effects. Recently, a transgenic mouse model has been produced that expresses active CK-Mit in liver (22). The CK-Mit localizes properly to the intermembrane space of mitochondria and is capable of synthesizing and utilizing PCr. These livers complement another set of transgenic mice that have been generated, which express the cytosolic CK-BB, enabling a direct comparison of changes in energy metabolism that may occur as a result of addition of different CK isoforms (18). An advantage of our approach of CK expression in the liver is the possibility of CK activation with dietary supplementation of Cr. Thus hepatic metabolism may be studied in the absence and presence of an active PCr circuit. In CK-BB-expressing livers no variation in free ADP levels was found over a wide range of enzyme activity and over a wide range of Cr levels (6). In the present work we used 31P NMR techniques to monitor the kinetics of CK in livers expressing CK-Mit and CK-BB. Contrary to a version of the shuttle hypothesis, CK-Mit catalyzes a reversible reaction, indicating that it is not perfectly coupled to the net flux of adenine nucleotides through the translocase. However, apparent free ADP levels drop with increasing activity of CK-Mit, even in the presence of CK-BB. These results indicate that CK-Mit can modulate mitochondrial function in vivo in an isoenzyme-specific manner.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation. Normal mice were a colony of inbred B6D2F1 (originally obtained from Taconic Farms, Germantown, NY). Transgenic mice expressing the human ubiquitous CK-Mit or rat brain CK-BB isoenzymes in their livers were created by incorporation of DNA fragments encoding for the proper CK isoenzyme under transcriptional control of the liver-specific transthyretin promoter as previously described (19, 22). Three homozygous lines of CK-Mit-expressing mice, Mit77, Mit33, and Mit11, expressed the enzyme at levels of 28, 483, and 560 µmol · g wet wt-1 · min-1, respectively. These mice were inbred with controls to generate heterozygotes for CK-Mit to further vary the level of enzyme expression. To create mice that were heterozygous for CK-BB and CK-Mit, homozygous lines of CK-BB and CK-Mit were inbred. These mice are referred to as CK-combined (CK-Comb) mice to indicate that they are expressing a combination of CK-BB and CK-Mit.

Mice were fed ad libitum with LiquiDiet (LD; Bio-Serv, Frenchtown, NJ) for 4 days before the experiments. Livers of normal and transgenic mice contain very low Cr concentrations (8). To increase intrahepatic Cr contents, the LD was supplemented with 1% or 5% Cr (Sigma, St. Louis, MO).

Mice were anesthetized with pentobarbital sodium (30 mg/kg ip; Sigma). The abdominal wall was opened by a midline incision below the xyphoid, and muscle and skin were reflected and sutured to the skin on the lateral abdominal aspects with 5-0 silk sutures. During anesthesia the mice were administered 0.9% saline-5% glucose solution intraperitoneally at doses of 2% body wt. After the experiments the mice were killed with an overdose of anesthetic (150 mg/kg pentobarbital sodium ip).

CK activity and isoenzyme distribution. CK activity (demonstrated by maximum PCr recovery rate, Vmax) was determined in homogenized tissue samples that were diluted 1:1,000 with an extraction buffer containing 26 mM Tris, 0.3 mM sucrose, 20 mM beta -mercaptoethanol and 1% tert-octophenyl poly(oxyethylene)ethanol (IGEPAL CA-630). All materials were obtained from Sigma. Total CK activity was determined spectrophotometrically at 37°C with a coupled hexokinase-glucose 6-phosphate dehydrogenase enzymatic system (procedure 45-UV; Sigma Diagnostics, St. Louis, MO). Diadenosine pentaphosphate (20 nM) was added to the reaction to inhibit adenylate kinase activity. Distribution of CK isoenzymes in CK-Comb mice was determined with Multitrac CK electrophoretic isoenzyme gels (Ciba-Corning, Marshfield, MA). Tissue homogenates were diluted 1:200 with the extraction buffer, and 1.0-µl samples were applied to the agarose gel. After color development, gels were scanned and relative intensities were determined as previously described (8).

NMR spectroscopy. Spectra were recorded with a Bruker 300-MHz spectrometer with 15-cm-wide horizontal bore magnet operating at 121.5 MHz for 31P. In vivo NMR spectra were collected with a 6-mm two-turn surface coil. Spectra were obtained by averaging 50 transients with a repetition time of 10 s. Free induction decays were multiplied with a 20-Hz line-broadening function, and peak integrals were measured with the Bruker integration subroutine. An external reference containing 4 µmol of methylphosphonic acid (MPA) and 12 µmol of gadolinium diethylenetriamine pentaacetic acid in 145 mM NaCl solution was used to compare phosphorus metabolites contents in each experimental group and in livers of transgenic and normal mice. An ATP concentration of 4 µmol/g wet wt was adopted from previous studies of livers of transgenic mice with HPLC and NMR to determine phosphorus metabolite concentrations (6, 7). Intracellular pH was measured from the chemical shift of Pi from PCr according to the relationship determined by Bailey et al. (3). Intracellular free magnesium concentration ([Mg2+]free) was estimated from the chemical shift of beta -ATP from alpha -ATP according to the relationship determined by Murphy et al. (25).

Saturation transfer experiments were performed to assess the pseudo-first-order rate constants for the transfer of phosphate from PCr to gamma -ATP [forward CK reaction to measure forward rate constant (kfor)] and from gamma -ATP to PCr [reverse CK reaction to measure reverse rate constant (krev)] (19). 31P NMR spectra for saturation transfer were acquisitions of 16 transients with a constant relaxation delay of 15 s followed by acquisition. Magnetization transfer was measured using low-power radio frequency (RF) pulses to selectively saturate gamma -ATP or PCr resonances for periods of 0.5, 1, 3, 5, 7, 9, and 15 s. These saturation pulses were applied on the resonance of gamma -ATP to determine kfor and on the resonance of PCr to determine krev. Symmetrical frequencies downfield from PCr and upfield from gamma -ATP were used to control for the effects of RF irradiation. The apparent longitudinal relaxation time (T1app) was determined from the monoexponential fit of the decay of magnetization as a function of the saturation time. Values of kfor and krev were calculated according to the equations 1/T1app = 1/T1 + k and k = (Mo - Mss)/Mo × T1app, where T1 is the spin-lattice relaxation time in the absence of exchange, Mo is magnetization observed without selective saturation, and the steady-state magnetization (Mss) was extrapolated from the exponential decay of signal at long saturation durations. Steady-state CK reaction velocities were obtained by multiplying metabolites concentrations with the rate constants: Vfor = kfor[PCr]; Vrev = krev[ATP].

Determination of Cr levels. Intrahepatic total Cr (Cr + PCr) contents were determined form livers after NMR experiments. Liver samples were diluted with 5 vols of 5% perchloric acid and homogenized. Samples were centrifuged at 10,000 g for 15 min at 4°C, and the supernatant was neutralized with KOH. Samples were centrifuged at 15,000 g for 15 min at 4°C. The pH of the supernatant was adjusted to pH 7.0 and lyophilized to dryness. Samples were dissolved in 2H2O containing 2 mM EGTA and 1 µmol of trimethylsylil propionic acid (TSP). 1H NMR spectra of tissue perchloric acid extracts were recorded on a Bruker 300-MHz NMR spectrometer operating at 300.13 MHz for 1H NMR. Spectra were acquisitions of eight transients with a repetition time of 8 s. Peaks were assigned and quantified using TSP as an internal reference. The methyl group of Cr + PCr was detected at 3.0 ± 0.1 ppm at pH 7.0 ± 0.6.

Calculation of free ADP levels. Apparent ADP levels were determined by assuming that the CK present in the liver catalyzed an equilibrium reaction. Because metabolite levels were in a steady state and no other reaction is known to produce or utilize PCr, this is a reasonable assumption. The expression ADP = ([ATP][Cr])/([PCr][H+]Keq), with an apparent equilibrium rate constant (Keq) = 1.58 × 109 at pH = 7.24 and [Mg2+]free = 0.65, was used (21). ATP levels were assumed to be 4.0 µmol/g wet wt. PCr levels were calculated from the PCr-to-ATP ratios determined from 31P NMR spectra, and Cr levels were determined from the difference in total Cr determined from tissue extracts by 1H NMR and the PCr levels determined from 31P NMR. ADP is reported in units of nanomoles per gram of wet weight and represents the sum of all ionic species of ADP.

Calculation of CK reaction rates. Enzyme flux was calculated with the equation derived to predict steady-state rates in reference to Vmax (18, 20): Vfor = (Vmax × [ADP] × [PCr])/[D × Km(ADP) × Kd(PCr)]. Vmax, [PCr], and [ADP] were measured as described above, and D is the sum of terms expressing metabolite concentrations divided by Kd and Km, the binary and ternary substrate-enzyme complexes, respectively, adopted from values reported for CK-Mit reaction in vitro (46).

CS and HK activities. Citrate synthase (CS) was extracted with a buffer containing 0.225 M mannitol and 0.075 M fructose in tissue homogenates. CS activity was measured spectrophotometrically at 37°C with a malate dehydrogenase-coupled reaction in a medium containing 10 mM oxaloacetate, 1 mM Ellman's reagent, 10 mM acetyl-CoA, and 10% Triton X-100 (29). Hexokinase (HK) was determined spectrophotometrically with a glucose-6-phosphate-coupled reaction in a medium containing 0.1 M Tris, 5 mM MgCl2, 2 mM glucose, 5 mM ATP, 0.5 mM NADP, 0.05% bovine serum albumin, and 0.5% Triton X-100 (29).

Tissue water contents. Tissue water (tw) contents were determined from the dry (dw) and wet (ww) weights of liver samples according to tw = (ww - dw)/dw. Dry weights were measured after 24 h at 85°C. Molar concentrations were calculated by assuming a ratio of intra- to extracellular water of 2/1 (28, 42).

Data analysis. All results are presented as means ± SD. Statistical significance was accepted at P < 0.05 by Student's t-test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mice expressing hepatic CK-Mit and CK-BB have been described previously (6, 8, 17, 18). It was possible to generate mice with a wide variety of CK-Mit activities by using the different lines of transgenic mice previously generated. Table 1 lists the different mice used, the genotypes, and Vmax CK activities determined in tissue extracts. Values represent means ± SD of groups of mice (n = 10). CK-Mit activity ranged from 28 to 560 µmol · g wet wt-1 · min-1 in five lines of mice. CK-BB activities were 920 ± 112 and 1,635 ± 172 µmol · g wet wt-1 · min-1 in the lines chosen for this study. Finally, CK-Comb mice were generated by interbreeding the homozygous CK-BB line with the homozygous Mit33 line. Total activity in CK-Comb was 1,135 ± 134 µmol · g wet wt-1 · min-1 with ~305 µmol · g wet wt-1 · min-1 (27%) due to CK-Mit and 830 µmol · g wet wt-1 · min-1 (73%) due to CK-BB.

                              
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Table 1.   Name, genotype, and creatine kinase activity for liver extracts of transgenic mice

31P NMR spectra indicated that a large amount of PCr was generated in livers expressing CK-BB and CK-Mit compared with control livers (Fig. 1). With the MPA standard, it was determined that ATP levels were not different among the different lines of transgenic mice (data not shown). To determine the in vivo rates of CK in the various livers, saturation transfer was performed. Fig. 2 shows representative spectra used to measure kfor from the CK-BB line of mice. In this case, saturation of gamma -ATP (Fig. 2B) led to a decrease in PCr compared with control irradiation (Fig. 2A) as clearly shown in the difference spectrum (Fig. 2C). Figure 3A shows that over the range of CK activities there was a linear relation between the CK rate measured in vivo with saturation transfer NMR and Vmax activities determined from extracts. The rate of CK measured in vivo was, in part, determined by Cr levels as shown in Fig. 3B. There was a linear increase in rate with increase in Cr. Detailed kinetic parameters are difficult to obtain in these experiments because PCr levels and ADP levels changed in addition to changes in Cr. However, the results obtained are in rough agreement with the in vitro measured values of Km of CK-BB and CK-Mit for Cr (46).


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Fig. 1.   31P NMR spectra from livers of normal mice (A) and mice expressing creatine kinase (CK)-Mit33 (B) and CK-BB (C) fed creatine. PCr, phosphocreatine; PME, phosphomonoesters.



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Fig. 2.   31P NMR spectra of a saturation transfer experiment in livers of transgenic mice expressing 1,635 units of the brain isoform (CK-B). Mice were fed 5% Cr for 4 days before the experiment. Each spectrum is a collection of 16 transients (90° pulses at a repetition time of 15 s). A narrow band saturation pulse (arrow) was applied at the frequency of gamma -ATP (B) and at a symmetrical control frequency (A) to determine magnetization transfer in the forward direction [phosphocreatine (PCr) right-arrow ATP]. C: difference between spectra A and B.



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Fig. 3.   A: measured CK forward flux [PCr right-arrow ATP; forward reaction velocity (Vfor)] from saturation transfer NMR as a function of Vmax CK activity determined in liver extracts (y = 22.4 + 0.3x; r2 = 0.95; P < 0.001) of mice expressing CK-B, CK-Mit, and both isoenzymes (CK-Comb). B: measured CK forward flux (PCr right-arrow ATP) from saturation transfer NMR as a function of liver creatine (y = -62 + 6.7x; r2 = 0.82; P < 0.05). gww, Gram wet weight.

Table 2 shows data obtained for kfor from all the mice and krev from some of the mice (n = 6 in each group). It can be clearly seen that CK-Mit mice with significant levels of CK catalyze a detectable reaction in both directions. If the forward rates of the CK reaction were equal to the reverse rates, the ratio of kfor/krev should be equal to the ATP-to-PCr ratios. This comparison is also shown in Table 2. For all of the mice evaluated there is good agreement between the ratio of metabolites and the ratio of rate constants, indicating that even though CK-Mit is localized to the inner mitochondrial membrane, it is able to catalyze its reaction at approximately equal rates in both directions.

                              
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Table 2.   Kinetic data obtained from saturation transfer NMR measurements of CK-expressing livers

Recently it was shown in vitro in hearts that the forward rate of the reaction catalyzed by CK-Mit can be adequately described by the enzyme's rate equation if the concentration of substrates is known (46). To test whether the measured and calculated concentrations of metabolites were consistent with the saturation transfer data, the measured forward rate of the CK-Mit catalyzed reaction ([PCr] × kfor) was compared with that predicted from the measured metabolites. Table 3 shows that over the full range of CK-Mit activities there is excellent agreement between the measured (n = 6) and predicted forward rates catalyzed by CK-Mit.

                              
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Table 3.   Measured and predicted forward rate catalyzed by the mitochondrial form of CK in transgenic mice expressing various levels of the enzyme

To check whether the presence of CK-Mit affected cellular energetics, free ADP levels were calculated in the various livers. Phosphorus spectra were collected in the presence of an external reference used to calibrate the peak integrals (Fig. 1). The apparent ATP contents of livers, within the experimental limitations of the surface coil, were stable in livers expressing various activities of both CK isoenzymes. Tissue PCr contents were determined by assuming an ATP concentration of 4 µmol/g wet wt. Total Cr (PCr + Cr) was determined from 1H NMR spectra of tissue extracts, enabling Cr levels to be calculated from the difference in total Cr and PCr levels. Table 4 presents values of Cr and PCr for livers of the different mice (n = 9). Free ADP levels were calculated by assuming equilibrium of the CK reaction and constant pH and [Mg2+]free. These values were similar in all the transgenic mouse livers with mean values of pH = 7.24 ± 0.04 and [Mg2+]free = 0.65 ± 0.06 mM (data not shown).

                              
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Table 4.   Metabolites and calculated ADP levels from transgenic mice expressing various levels of CK-Mit and CK-BB

Interestingly, there is a variation in ADP levels with activity of CK-Mit. Calculated ADP concentrations are plotted against total CK activity in Fig. 4. The calculated ADP concentrations decreased as a function of Vmax in livers expressing CK-Mit. Calculated ADP levels fell from 84 nmol/g wet wt in Mit77 mice to 38 nmol/g wet wt in Mit11 mice. Decreasing the total Cr by reducing dietary Cr had no significant effects on the calculated ADP levels. Assuming an intracellular water-to-extracellular water ratio of 2/1, molar concentrations of ADP ranged from 128 to 54 µM. The differential uptake of Cr in livers with various CK activities may have increased the ratio of intra- to extracellular water, resulting in overestimation of the molar concentrations of ADP. Mice expressing two levels of CK-BB had intermediate ADP levels of ~52 nmol/g wet wt (78 µM). Previous results varying CK-BB activity over a wide range did not detect differences in calculated ADP (6), consistent with values similar to those found here over a twofold range of CK-BB activity. Interestingly, livers expressing both CK-BB and CK-Mit had low levels of ADP consistent with the levels determined from livers expressing CK-Mit only.


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Fig. 4.   Hepatic ADP levels calculated from in vivo 31P NMR measurements vs. CK activity determined in vitro in livers of transgenic mice expressing CK-B, CK-Mit, and both isoenzymes (CK-Comb). Mice were fed 5% Cr for 4 days before the experiment. open circle , Groups of CK-Mit mice fed 1% Cr. ADP concentration decreased as an exponential function of CK activity in all mice expressing CK-Mit [y = 36 + exp(-x/220); r2 = 0.96; P < 0.001].

The relatively large changes in ADP associated with expression of CK-Mit in the liver might be expected to cause adaptation of liver metabolism. To begin to address this issue, levels of CS and HK were measured (n = 6) in control and CK-Mit-expressing livers (Table 5). After 4 days of 5% Cr feeding, CS activity tended to decrease with increasing activities of CK-Mit (y = 131 - 0.07x; r2 = 0.74; P < 0.05). A similar change was observed in HK activity as CK-Mit activity increased (y = 2.2 - 0.0001x; r2 = 0.96; P < 0.005). These results indicate that the calculated change in ADP has functional consequences for hepatic metabolic capacities.

                              
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Table 5.   Effects of varying activities of mitochondrial CK on citrate synthase and hexokinase activities in transgenic mice fed 5% creatine for 4 days


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present work makes two important contributions to the understanding of the function of CK-Mit. The first contribution is the demonstration that CK-Mit can cause a decrease in apparent levels of ADP. ADP concentrations calculated assuming CK equilibrium ranged from as high as 84 ± 9 nmol/g wet wt in livers with low levels of CK-Mit to as low as 38 ± 4 nmol/g wet wt in livers with the highest levels of CK-Mit. This calculation was based on whole liver measured values of the indicator metabolites PCr, Cr, ATP, pH, and Mg2+. A number of arguments indicate that ADP levels were lowered by functional incorporation of CK-Mit in livers of the transgenic mice. First, the predicted fluxes for CK-Mit based on the measured metabolites matched the measured values very closely (Table 3). Second, ADP concentrations were similar at different dietary Cr concentrations, indicating adjustment of indicator metabolite concentrations according to the CK equilibrium (Table 4). Third, in mice expressing both CK-Mit and CK-B (CK-Comb mice) there was a significant decrease in apparent ADP levels (Table 4). Evidently CK-Mit was the dominant factor in determination of ADP concentration in livers of CK-Comb mice, within the range of ADP levels in CK-Mit mice. Fourth, there were decreases in measured levels of citrate synthase and hexokinase, indicating that the liver was adapting to a change in energetics, as might be expected if ADP levels were lower in the livers expressing CK-Mit (Table 5).

The results comparing calculated ADP levels in the various mice open the question of what the free ADP level is in a normal liver. 31P NMR estimates put it at ~200 nmol/g wet wt (11, 12, 16). These results are difficult because of the low levels for NMR sensitivities and the fact that differences between peaks must be monitored (43). Assuming that glycolysis maintains equilibrium through three steps, a value of free ADP of 50 nmol/g wet wt has been reported (47). This work relied on determination of a number of indicator metabolites, and it is likely that levels of free Pi were overestimated, leading to an underestimate of free ADP levels. In our own work expressing CK-BB in liver, we have measured free ADP levels of ~50 nmol/g wet wt over a wide range of CK activity and Cr concentration (6, 18). However, in these studies the lowest enzyme activities were higher than the highest CK-Mit activities in the present work, indicating that any decreases in ADP caused by CK-BB may have been saturated even at the lowest activities. The high level of ADP (84 ± 9 nmol/g wet wt) obtained in the lowest CK-Mit-expressing livers indicates that both CK-B and CK-Mit have lowered hepatic ADP levels compared with normal livers. Indeed, extrapolation of the apparent ADP levels to zero CK activity gives values of ~135 µM, consistent with the NMR results from normal livers (11, 12, 16). Thus it may be that both CK-B and CK-Mit decrease hepatic ADP levels; however, it is clear that CK-Mit is able to maintain lower levels of ADP than CK-B.

There are a number of possible mechanisms whereby CK-Mit could cause decreases in ADP levels in liver. Most in vitro work demonstrates that the presence of CK-Mit causes a decrease in the Km of ADP for oxidative phosphorylation (unpublished data). No significant differences in oxygen consumption rates have been measured in perfused livers expressing CK-Mit compared with normal livers (unpublished data). Assuming that rates of oxidative phosphorylation are the same in transgenic livers expressing varying levels of CK-Mit and CK-B, these rates can be supported by lower levels of ADP in livers with high levels of CK-Mit, thus enabling them to maintain lower ADP levels. These findings corroborate previous observations that mitochondria isolated from mice expressing CK-Mit had higher rates of respiration at submaximal levels of ADP (27). Consistent with the notion that CK-Mit can modulate the Km of oxidative phosphorylation for ADP is the finding that increases in cardiac work cause larger changes in ADP in hearts lacking CK-Mit and CK-MM for a similar change in oxygen consumption (36). Interestingly, levels of ADP were the same in hearts lacking CK-Mit and CK-MM compared with control at low levels of work (36, 48). Experiments that use fructose to increase hepatic ADP and oxygen consumption should enable direct quantification of the Km of ADP for oxidative phosphorylation in transgenic livers (7).

There are possibilities for how CK-Mit might lower ADP levels other than affecting the relation between oxidative phosphorylation and ADP. Recently, imaging studies of single mitochondria indicate that there is spontaneous depolarization of mitochondria caused by opening of the mitochondrial permeability transition pore (MPT; Ref. 15). There is evidence that the MPT is the ANT. If liver mitochondria are spontaneously depolarizing at a significant rate one would expect higher levels of ADP than if they depolarized at a slower rate. Recent studies on mitochondria isolated from CK-Mit-expressing livers indicates that CK-Mit can inhibit the MPT, which in turn could lead to lower levels of cellular ADP (27).

Finally, it may be that substrate utilization by mitochondria is affected by CK-Mit. Tissues with high levels of CK-Mit, such as heart, tend to rely on fatty acids as the primary substrate for oxidative phosphorylation, whereas tissues with low levels of CK-Mit, such as brain or skeletal muscle, tend to rely on carbohydrates. Although it is difficult to imagine a direct connection between CK-Mit levels and mitochondrial substrate utilization, it may be that changes in ADP levels alter substrate utilization, which can in turn affect ADP levels. Preliminary results indicate that mouse livers expressing CK-Mit are more sensitive to inhibitors of mitochondrial fatty acid transport than normal or CK-B-expressing livers (unpublished observation).

The other important finding of the present work is that 31P NMR rate measurements of CK isoenzymes expressed in transgenic livers indicate that the net forward fluxes were approximately equal to the reverse fluxes. This result was expected in steady-state metabolic conditions in livers expressing one of the isoenzymes, indicating catalysis of Pi transfer between ADP and Cr at equal rates. We also measured equal fluxes in CK-Comb livers expressing both CK isoenzymes. Reversible Pi transfer at equal rates was demonstrated in vitro (46), with ex vivo stimulation of slow twitch muscle (22) and in vivo during a maturational increase in CK activity in the mouse brain (14). However, other in vivo studies have reported Vfor-to-Vrev ratios of 2-2.4 in tissues with variable composition of CK isoenzymes, including brain, heart, and skeletal muscle (5, 26, 39, 45). The experimental explanation given to this phenomenon was incomplete detection of the exchange of NMR-invisible metabolites because of intracellular compartmentation (19) and participation of ATP in multiple phosphorus transfer reactions, whereas PCr transfers its high-energy phosphate solely to ATP (45). Both explanations could be true in the case of the liver under baseline metabolic conditions. In the liver equal forward and reverse rates of saturation transfer may result from absolute NMR visibility of phosphorus metabolites, compartmentation of equal amounts of substrates, or exchange rates from ATP to PCr faster than the competing reactions.

The PCr shuttle hypothesis postulates that cytosolic and mitochondrial CK isoenzymes form a close circuit of ATP synthesis in the cytosol and hydrolysis in mitochondrial intermembrane space (1, 24, 32, 34, 50). This model would predict a much higher rate of CK-Mit in the direction of ATP + Cr right-arrow ADP + PCr. Considering that the outer mitochondrial membrane leaflet is relatively impermeable to ADP (33), ADP concentrations in mitochondrial intermembrane space are higher (32) and CK-Mit is functionally coupled to the ANT (24, 34, 50). According to this model, the flux through CK-Mit should be equal or proportional to that through the ANT, attributing a transport function to CK-Mit in addition to its energy buffering activity in excitable tissues (1, 50). If the main role of CK-Mit is to recycle nucleotides coupled to ANT activity in mitochondrial intermembrane space, flux in the ADP-generating direction (Vrev) should be higher than the forward flux (Vfor).

We have measured roughly equal net fluxes in the forward and reverse directions over a wide range of CK-Mit activities, as expected from an enzyme operating at steady state. The measured CK fluxes could be predicted from the rate equation using the concentrations of participating metabolites, and there was a roughly linear relationship between the reaction velocity and total CK activity irrespective of the isoenzymes expressed by the livers. These findings indicate thermodynamic regulation of hepatic CK reaction velocity in the transgenic mice, lacking any vectorial predilection. Furthermore, in perfused livers the flux through CK-Mit was steady when oxygen consumption was increased by phenylephrine (unpublished data). This evidence argues against a strong coupling between CK-Mit and ANT that would create unequal net fluxes.

One may suggest that in the transgenic livers the human ubiquitous CK-Mit was not able to pair with the mouse ANT to form a strongly coupled system. We have evidence that in the transgenic livers ~93% of CK-Mit is associated with the mitochondrial membrane (23) and ADP concentrations are modulated by this isoenzyme in vivo. Furthermore, the presence of CK-Mit in the liver alters the sensitivity of mitochondrial respiration to changes in ADP and substrate preferences (unpublished data). Therefore, it is clear that CK-Mit catalyzes roughly equal forward and reverse rates of the reaction even though it is confined to the intermembrane space. We were unable to determine oxygen consumption in vivo; therefore, the flux through CK-Mit could be not related to ANT activity at various work loads.

In conclusion, expression of CK-Mit in transgenic livers led to an activity-dependent decrease in calculated ADP levels. This decrease in ADP occurred even in the presence of CK-B. This represents in vivo evidence that CK-Mit can have specific effects on cellular energetics. This CK-Mit effect may explain the recently observed effects of CK-Mit on cellular growth (2).


    ACKNOWLEDGEMENTS

The authors acknowledge Dr. Virgil Simplaceanu for assistance with NMR experiments and Kathy Sharer for assistance with the mice.


    FOOTNOTES

This work was supported by a National Heart, Lung, and Blood Institute (NHLBI) research career development award to A. P. Koretsky (HL-02847), NHLBI Grant R01-HL-40354, and a postdoctoral fellowship from the Pennsylvania Affiliate of the American Heart Association to N. Askenasy.

Address for reprint requests and other correspondence: N. Askenasy, Box 164, Mellon Institute, 4400 Fifth Ave, Pittsburgh, PA 15213 (E-mail: askenasy+{at}andrew.cmu.edu).

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.

First published October 3, 2001; 10.1152/ajpcell.00404.2001

Received 17 August 2001; accepted in final form 1 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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