A possible role of inorganic phosphate as a regulator of oxidative phosphorylation in combined urea synthesis and gluconeogenesis in perfused rat liver. A phosphorus magnetic resonance spectroscopy study

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candidate as a regulator of mitochondrial respiration in vivo with two exceptions of the phosphofructokinase-deficient limb (9) and the fructose-loaded liver (10). Comparisons of Pi value in liver as measured by NMR and biochemical analysis have shown that free Pi is approximately one-half of the total Pi (11,12). Therefore, the role of free Pi in regulation of oxidative metabolism should be reconsidered. The free form of ADP is also differentiated from the bound form based on the difference of spin-lattice (TI) relaxation time. However, in vivo studies on the role of free ADP and free Pi in the metabolic control of the liver has been performed.
A small decrease of ATP in perfused rat liver during the operation of a biosynthetic pathway such as gluconeogenesis has been reported by several investigators using analytical biochemistry (13, 14). Phosphorus magnetic resonance spectroscopy enables consecutive determination of small changes in ATP and Pi in the same liver. The @ATP peak in phosphorus spectra in perfused rat liver stands independently and is easy to quantitate. However, the Pi peak is difficult to separate from those of phosphomonoester and phosphodiester. Liver as opposed to brain and muscle lacks the phosphocreatine/creatine energy buffering system. Therefore, the changes in Pi are attributable directly to ATP breakdown, if no sugar phosphate accumulates and no phosphate is lost from hepatocyte. ADP is also produced in an equal amount by ATP breakdown. Thus, the K,,, value of the relation between oxygen consumption rate (A$')o, and changes in ATP concentration (-AATP) will provide a clue as to metabolic control based upon a 20 times difference between the K,,, value in mitochondria for ADP and the value for Pi.

EXPERIMENTAL PROCEDURES
Materials-Male Sprague-Dawley rats, weighing 120-140 g, were fed laboratory chow ad libitum. Food was withdrawn 48 h before the start of the perfusion experiment. The animal had free access to water at all times. All chemicals were purchased from Sigma. Enzymes were obtained from Boehringer Mannheim.
Liver Perfusion-After anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight), the liver was perfused with phosphate-free Krebs-Henseleit solution with 0.5 mM EDTA equilibrated with 95% 0 2 and 5% CO, following the method described by Sugano et al. (15). The excised liver was placed in a closed plastic cylinder with 30 mm diameter and 22 rnl volume inside of a horizontally placed solenoid coil. A high perfusion flow rate of 27 m1/100 g body weight ensured sufficient oxygen supply with hemoglobin-free perfusion. The perfusate entered the liver at a constant temperature of 35 "C controlled by a water-jacketed line, and the pH of the perfusate was 7.35. The effluent from liver was collected by two suction lines. One was positioned at the bottom of the capsule to measure oxygen partial pressure without contamination of air. Oxygen partial pressure was recorded polarographically with a Clark-type oxygen electrode placed 50 cm distant from the NMR capsule. The oxygen consumption rate was calculated from the flow rate, and the difference in the oxygen partial pressure between the influent and the effluent using the Bunsen absorption coefficient in crystalloid perfusate was 3.25 ml Oz/ml/mm Hg 0 2 (16). Urea and glucose production rates were determined with the sample collected from this line. The second suction line was placed at the top of the capsule at a flow rate of more than 50 ml/min and allowed the liver to bathe in the perfusion medium and prevented overflow outside of the NMR capsule.
Experimental Protocol-The liver was challenged by four levels of combined urea synthesis and gluconeogenesis by changing the perfusate containing these substrates. After recording two control phosphorus spectra without the addition of substrates, 5 mM lactate and 1 mM pyruvate were added to initiate gluconeogenesis. 20 minutes after the initiation of gluconeogenesis, the following four levels of substrates for urea production were further added for 20 min in the following four groups: 0.5 mM NH&l in Groups 1, 2 mM NH4Cl in Group 2, 2 mM NH&l and 2 mM ornithine in Group 3, and 10 mM NH,Cl and 2 mM ornithine in Group 4, respectively. The 20-min interval was allowed to reach steady state. At 5-min intervals, phosphorus spectrum, oxygen consumption rate, urea, and glucose production rates were measured. At the end of the experiment, the perfused liver was freeze-clamped with aluminum tongs precooled in liquid nitrogen for the biochemical measurement of adenine nucleotides and Pi. The first control spectrum was measured at 30 min, and within 80 min the entire experiment was finished. In the control group the liver was perfused without additional substrates, and phosphorus spectra and oxygen consumption were measured at three periods: 30-40, 50-60, 70-80 min. The liver sample freeze-clamped at 30 min after initiation of perfusion without substrates served as control for the biochemical analysis.
NMR Measurement-Phosphorus spectra were obtained at 5 tesla on a Brucker CXP 200 with a four-turn solenoid type probe tuned to 80.98 MHz. Spectra of perfused liver were obtained typically for 4.2 min by accumulating 512 free induction decays resulting from 47-ps radiofrequency pulses. The angle of the spin was go", and the delay between each pulse was 0.5 s. The repetition rate and the flip angle were selected to optimize the time resolution of 0-ATP (17). Fourier transformation was performed with 20-Hz exponential line broadening. Dimethyl phosphonic acid in a capillary in the perfusion chamber was used as an external reference to standardize changes in signal intensity (11). Relative concentration of ATP, Pi, and phosphomonoester were calculated by triangulation (height versus width at halfheight) from &ATP, Pi, and phosphomonoester peak area. ADP concentration was estimated by measuring the difference in the area under the ?-ATP + @ADP and the @ATP peaks. The Pi/ATP and ADP/ATP ratios were corrected for saturation. In this study, the saturation parameters of Pi, @-ATP, and y-ATP were 51.0 f 2.0, 33.3 to-noise ratio of @-ATP was around 20-30. Intracellular pH was f 1.2, and 31.0 f 1.7% (n = 3, mean f S.D.), respectively. The signalcalculated from the Pi chemical shift relative to a-ATP according to the equation described by Malloy et al. (18).
Biochemical Analysis-Biochemical analysis of ATP, ADP, AMP, and Pi concentration was performed in neutralized HClO, liver extracts (19)(20)(21). The concentrations expressed as micromoles per gram wet weight of liver were converted to millimolar using the value of 0.8 g of cellular HzO/g of liver (22). Urea and glucose were measured by enzymatic spectrophotometric methods described in Refs. 23 and 24. Statistical Analysis-The data were analyzed using an analysis of variance and unpaired Student's t test on the four groups versus control with the confidence limit at 95%. The values are expressed as mean f S.D.

RESULTS
Four levels of oxygen consumption rate were obtained by substrate loading for urea synthesis and gluconeogenesis, as shown in Fig. lA and Table I. Combined urea synthesis and gluconeogenesis caused a decrease in ATP concentration in response to the four substrate combinations, as shown in Fig.   1B and Table 11. Within 15 min after the addition of substrates, oxygen consumption, urea and glucose production rate, and ATP level reached steady state. In the control group without substrate loading, ATP and Pi concentration remained at the initial level at least for 80 min of perfusion. Typical spectra before and during combined gluconeogenesis and urea synthesis and the difference spectrum are shown in Fig. 2.

FIG. 2.
Phosphorus spectra before and during combined urea synthesis and gluconeogenesis with 10 mM NHdCI, 2 mM ornithine, 5 m~ lactate, and 1 mM pyruvate and the difference spectrum between them. Trace a shows the control spectrum, Trace b shows the spectrum during combined urea synthesis and gluconeogenesis, and Trace c shows the difference spectrum between a and b. A typical case is shown. Assignment: 1, reference; 2, phosphomonoester; 3, Pi; 4, ?.-ATP + 6-ADP; 5, a-ATP + a-ADP; 6, 6-ATP.
the control was 2.51 f 0.20 and 3.24 f 0.26 mM as shown in Table 111. The Pi/ATP ratio measured by analytical biochemistry in the control was 1.34 f 0.22, while the ratio by NMR was 0.81 f 0.20. Assuming the 100% visibility of ATP, comparison of these values revealed that NMR-visible Pi is approximately 60% of Pi determined by biochemical analysis, that is 1.94 mM, as summarized in Table IV. The comparison of ADP/ATP ratio by NMR and biochemical analysis in control showed that NMR-visible ADP is approximately 20% of ADP measured by enzymatic analysis, that is 0.20 mM. Combination of urea synthesis and gluconeogenesis evoked significant decrease in ATP by 4.6, 10.8, 15.3, and 23.8% (0.12, 0.27, 0.38, and 0.60 mM) relative to each control in the four groups, respectively, while NMR-visible Pi concentration was significantly increased in Group 3 and Group 4 by 15.8 and 26.9% (0.31 and 0.52 mM), as shown in Table 11. Gluconeogenesis alone did not change ATP or Pi concentration. Table V shows that total values of (ATP + NMR-visible Pi) of 4.36, 4.33, 4.38, and 4.37 mM in the four groups were maintained almost at the same level as the control of 4.45 mM. Analytical biochemistry showed that Pi concentration in the effluent was under the detectable level both in the presence and absence of substrates at least for 80 min. Phosphomonoester peak area, which contains phosphorylcholine, sugar phosphate, AMP, and 3-phosphoglycerate (25), did not significantly change during this substrate loading, as shown in Table 11. ATP and Pi concentration in control remained at the initial level at least for 80 min. These results suggest that there is a stoichiometric relationship between an increase in NMR-visible Pi and the decrease in ATP.
Intracellular pH was significantly decreased both during gluconeogenesis alone and during combined urea synthesis and gluconeogenesis as shown in Table 11. However, the values of oxygen partial pressure in the effluent during combined urea and glucose synthesis were 287 f 54,272 f 63,228 f 63, 175 f 45 mm Hg (n = 6) in the four groups which strongly suggests that sufficient oxygen was supplied to liver.

Control
Group 1  Significantly different from the control ( p < 0.05).

TABLE V The stoichiometric relationship between decrease in ATP and increase in free P i during combined urea synthesis and gluconeogenesis
Group 1, 5 m M lactate + 1 mM pyruvate + 0.5 mM NH4Cl; Group 2, 5 mM lactate + 1 mM pyruvate + 2 mM NH4CI; Group 3, 5 mM lactate + 1 mM pyruvate + 2 mM NH&l + 2 mM ornithine; Group 4, 5 mM lactate + 1 mM pyruvate + 10 mM NH4C1 + 2 mM ornithine; between the (7-ATP + P-ADP) and ,&ATP area in the 31P NMR spectrum. The NMR-measured relative ratio of ((7-ATP + @-ADP) -6-ATP) to P-ATP has been reported to be in the wide range of 0.06-0. 14 (11, 12, 25). In the present study, the NMR-measured ADP/ATP ratio in control was 0.08 f 0.04 (n = 30) with large standard deviation. Table VI shows the changes in calculated NMR-visible ADP concentration during combined urea synthesis and gluconeogenesis. The calculated NMR-visible ADP concentration was not significantly changed from the control value of 200 PM in response to the four levels of substrate loading. However, the
ATP was calculated according to the following equation:  Table 111. The changes in the Pi concentration were not significant. With biochemical assay total ADP concentration was increased from the control value of 0.98 f 0.11 to 1.14 f 0.15 mM in Group 4.

DISCUSSION
Decrease in ATP-ATP synthesis should be equal to ATP consumption in the steady state metabolism. Initial depletion of ATP was caused by combination of urea synthesis and gluconeogenesis due to lack of energy buffering systems such as phosphocreatine/creatine which exist in brain and muscle but not in liver. The decrease in ATP is induced by fructose infusion or massive hepatectomy (26,27). Fructose is rapidly phosphorylated to fructose 1-phosphate by ketohexokinase at the expense of ATP, resulting in depletion of ATP and sequestration of Pi. Since liver regeneration requires increased ATP synthesis due to protein and nucleic acid synthesis, the concentration of ATP in the remnant rabbit liver 24 h after 70% hepatectomy is decreased by 40%. Gluconeogenesis alone did not significantly affect the ATP level in the present study. Lactate and pyruvate act both as substrates for gluconeogenesis and as more effective energyyielding substrates than endogenous substrates (14). Conversely, ammonium chloride is solely a metabolic load to liver from the viewpoint of energy balance. Ammonia arises almost exclusively from protein breakdown, and the concentration of

Metabolic Control
of Rat Liver ammonia in the portal vein reaches 1.0 mM in rats fed with high protein food (28). Hyperammonemia is known to deteriorate liver function and is considered as one of the causes of hepatic failure. Although the rapid perturbation by combination of gluconeogenesis and urea synthesis with 10 mM NH&l and 2 mM ornithine plus 5 mM lactate and 1 mM pyruvate is far from a physiological condition, a depletion of adenine dinucleotides was not observed. The decrease in ATP during the perturbation could not be ascribed to deterioration of the liver judging from time course changes in the control group without substrate loading.
Urea synthesis from ammonium chloride and gluconeogenesis from lactate and pyruvate require four ATP per urea, six ATP per glucose, respectively. Urea and glucose production need one oxygen per glucose and two-third oxygen per urea, assuming the P/O ratio to be 3. The balance between oxygen consumption and glucose and urea synthesis may be estimated by the following equation: AVo,/(AG + 2 AU/3), where AVoz is the difference of oxygen consumption rate between the accelerated and the control value, AU and AG are net production of urea and glucose. The calculated values are 1.14, 1.36, 1.05, and 1.04 in Groups 1 , 2 , 3 , and 4, as shown in Table VII. The values over 1 suggest that the small decrease in ATP is not due to hypoxia. The values of oxygen partial pressure in the effluent showed that relative hypoxia in the pericentral area could be avoided by the high perfusion flow rate. The observed slight decrease in intracellular pH might be due to urea synthesis itself which partially involves carbonic anhydrase reaction and produces protons (29).
Pi Control and ADP Control-NMR-visible Pi and ADP are "free" and are likely to participate in mitochondrial oxidative phosphorylation. Invisible Pi and ADP are "bound." Based on both the present NMR study and enzymatic analysis, the concentrations of ATP and free cytosolic Pi (Pi,) in the control were estimated to be 2.51 and 1.94 mM. The controlling chemical of oxidative metabolism must be in the range of the K , value. Since free Pi concentration in hepatocyte is close to the reported K , value for respiration of 1 mM, Pi is considered to be one of the regulators. Fig. 3 shows the kinetic relationship between AVO, (AVO, = VoZ accelerated -Vo, at rest) and decrease in ATP (-AATP). Oxygen consumption ( VO,) includes all ATP-consuming processes and cyanide-insensitive respiration. Even at the resting state ATP is utilized continuously for maintenance of cell integrity and function using endogenous substrates. Therefore, AVO, and AATP are net oxygen consump-

TABLE VI1
The relative ratio of AVQ to gluconeogenesis and urea synthesis Group 1, 5 mM lactate + 1 mM pyruvate + 0.5 mM NH4Cl; Group 2, 5 mM lactate + 1 mM pyruvate + 2 mM NH4C1; Group 3, 5 mM lactate + 1 mM pyruvate + 2 mM NH,Cl + 2 mM ornithine; Group 4, 5 mM lactate + 1 mM pyruvate + 10 mM NH4Cl + 2 mM ornithine.  where AVO, is pmol/min/g liver and -AATP is mM. This equation was fitted according to nonlinear least squares regression. This implies that K, value of Avoz for -AATP is 0.35 mM. Pi and ADP are theoretically produced in equal amounts by ATP breakdown. The K,,, value for Pi was reported to be 1 mM, while the K,,, value for ADP was reported to be 20 p~. The observed high K,,, value of 0.35 mM favors that respiratory activity associated with combined urea synthesis and gluconeogenesis is regulated by changes in Pi, since 50fold smaller changes of ATP would be expected if ADP were in control of liver metabolism. The decrease in ATP can be substituted for the increase in Pi based on the stoichiometric relationship between them. Therefore, free Pi can be estimated as the following equation: Free Pi (Pit) = 1.94 + (-AATP) (mM) Chemical reaction velocity does not respond to a differential change in substrate but to the molar concentration of substrate. Fig. 4 shows the kinetic relationship of oxygen con-sumption as a function of calculated free Pi. where Pi, is in mM. The result suggests that 0.24 mM increase of free Pi above a calculated base-line value of 1.81 mM causes half-maximal acceleration of oxidative metabolism and that Pi available for mitochondrial phosphorylation may be rather low. The reported K,,, value of oxidative phosphorylation for Pi in isolated mitochondria varied with animal species and substrates. The value of rat liver mitochondria was reported as 1 mM in the presence of /.?-hydroxybutyrate (51, while the value of pigeon heart mitochondria was 0.13 mM in the presence of glutamate and succinate (30). The observed "K,,," value of 0.24 mM is thus close to the reported K,,, value for Pi of isolated mitochondria.
Pi content in rat liver has been reported to be in the wide range of 2.5-5.2 pmol/g wet liver. Hansen et al. (31) and Quistorff and Poulsen (32) claimed their low value of 2.4-2.9 pmollg wet weight to be unbiased by sampling and contraction artifacts and explained the discrepancy due to the possibility of hydrolysis of labile organophosphate compounds (31, 32). Cunningham et d . (12) have reported that the NMR visibility of P, is about 50% under various conditions (12). In the present study, Pi concentration in the control as measured by biochemical analysis was 2.59 pmol/g wet weight and NMR visible Pi represents about 60% of total Pi. The present study revealed at the organ level that Pi may be a controller of liver mitochondrial oxidative metabolism based on the kinetic relationship. Chance and Hess (33) have found that transient activation of respiration by glucose addition in ascites tumor cell is characterized by a high ADP level and that the inhibition of respiration by glycolysis is related to the intracellular levels of phosphate. Wu and Racker (34) have reported in the study of the Pasteur effect that K,,, value for Pi of glyceraldehyde-3-phosphate dehydrogenase of ascites tumor was 1.5 mM, while the intracellular concentration of Pi was 3-9 mM. The difference was interpreted by compartmentation and sequestration of Pi by organelles. Chance and Maitra (35) have found that only a small portion of the total phosphate in the cell is available to participate in the phosphate potential based on the difference between the spectroscopic method and chemical determination.
It should be noted that phosphate was omitted in the perfusion medium to evaluate the changes in intracellular Pi (25). Leakage of Pi from hepatocytes into the perfusate was negligible. Thus, NMR did not measure Pi in the perfusate in the capsule but Pi in hepatocytes. Both biochemical and NMR studies showed that Pi was increased by combined urea synthesis and gluconeogenesis. The urea synthesis rate was comparable to other reports using ordinary Krebs-Henseleit solution (14,28,29). These results eliminate the possibility that lack of phosphate in the medium requires Pi to be a regulator in oxidative phosphorylation.
ADP are largely bound as opposed to ATP and the free ADP has been proposed as a regulator of oxidative phosphorylation in muscle and brain, since the free ADP concentration calculated by the equilibrium constant of the near equilibrium reaction catalyzed by creatine kinase is in the range of K,,, value (7, 8, 36). However, creatine kinase system is not expressed in the liver. The NMR measurement of ADP gives directly the free ADP concentration using the difference between (7-ATP + /.?-ADP) and @-ATP, although the sensitivity of quantification of ADP by organ magnetic resonance spectroscopy should be considered (12). NMR-calculated free ADP concentration in the perfused rat liver was reported to be in the range of 176-300 p~ (11,12,25). Veech et al. (22) calculated the cytosolic free ADP in hepatocytes as 46 p M from the measured components of glyceraldehyde-3-phosphate dehydrogenase:3-phosphoglycerate kinase/lactate dehydrogenase reactions. In the present study NMR measurement revealed that free ADP concentration in control was around 200 p~. Free ADP concentration exceeds the range of the K,,, value. However, the NMR study showed that calculated free ADP concentration was increased in response to the perturbation by 10 p~ with a large standard deviation. It might be suggested that a large part of ADP from ATP breakdown becomes the bound form. The reported 20 times difference between the K,,, value for Pi and that for ADP might be explained by the difference of binding capability. The contribution of free ADP in the respiratory control mechanism remains unclear since the in uiuo K,,, value for free ADP could not be determined in this study. Ylikahri et ~l . (10) reported that fructose infusion to rat liver induced transient inhibition of oxidative metabolism concomitant with decreases in Pi and ATP, as fructose is metabolized at a high rate via glycolysis. Pi was decreased by fructose infusion from 3.9 to 1.9 mM, which value is nearly equal to K,,, value for P, in oxidative phosphorylation. Moreover, they reported that mitochondrial oxygen uptake rates in mammals are related to plasma Pi concentration (37). However, systematic analysis for K,,, value is necessary to verify the control mechanism of oxidative metabolism in liver. The present systematic analysis of oxidative metabolism during urea synthesis and gluconeogenesis has revealed that oxidative metabolism may be controlled by feedback of increased Pi.