The Journal of Nutrition Vol. 128 No. 1 January 1998, pp. 28-34

pH Is Regulated Differently by Glucose in Skeletal Muscle from Fed and Starved Rats: A Study Using ³¹P-NMR Spectroscopy^1,2

Dominique Meynial-Denis^{*, 3}, Michèle Mignon^*, Loïc Foucat, Guy Bielicki, Ahmed Ouali^**, Caroline Tassy^**, Jean-Pierre Renou, Jean Grizard^*, and Maurice Arnal^*

^* Unité d'Etude du Metabolisme Azoté et Centre de Recherches en Nutrition Humaine d'Auvergne;  Unité STIM-SRV; and ^** Unité Métabolisme Energétique-SRV, INRA Theix Centre de Recherches de Clermont-Ferrand-Theix-63122-Ceyrat, France

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The aim of this study was to determine whether exogenous glucose metabolism influences the pH in superfused EDL muscle from growing rats fed or starved for 48 h (body weight 55 and 45 g, respectively). Energy state and intracellular pH of muscle were repeatedly monitored by ³¹P-nuclear magnetic resonance spectroscopy (³¹P-NMRS); glycogen and other energy metabolites were assayed enzymatically in muscle extracts at the end of the experiment. In EDL muscles from starved rats superfused with glucose for 4 h, intracellular pH was elevated (7-7.3), lactate concentration low, glycogen repletion very intense and citrate synthase activity high. We conclude that glucose was routed mainly toward both oxidative phosphorylation and glycogen synthesis in EDL muscles after food deprivation of rats. In contrast, the major pathway in muscles from fed rats may be glycolysis because the glycogen pool remained constant throughout the experiment. The additional and minor pH component (in the range of 6.5 to 6.8) seen in muscles from fed rats, even in the presence of exogenous glucose, might be due to impaired glucose utilization because this component appears also in muscles from starved rats superfused without glucose or with a nonmetabolizable analog of glucose. Consequently, direct pH measurement by ³¹P-NMR may be considered to be a precise criterion for evaluation of differences in metabolic potentialities of muscle studied ex vivo in relation to the nutritional state of rats.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Intracellular pH is tightly regulated in cell metabolism (Frelin et al. 1988, Madshus 1988, Roos and Boron 1982). Numerous studies have demonstrated marked changes in its value in pathophysiologic conditions. For example, resting intramuscular pH is more acidic in streptozotocin-diabetic rats (Challiss et al. 1990) and more alkaline in dystrophic mdx mice (Dunn et al. 1992) than in controls. These pH anomalies were either maintained or abolished during exercise. Exercise induces two pH compartments with a high and low pH in both healthy men and dystrophic dogs. This compartmentation was related to different recruitment of oxidative and glycolytic fibers (Achten et al. 1990, Mc Cully et al. 1992, Mizuno et al. 1994, Park et al. 1987). In resting extensor digitorum longus (EDL)³ muscles ex vivo, an important pH heterogeneity can appear if the biochemical and physiologic stability is compromised by an impaired O₂ diffusion due to a greater thickness of muscle. In this case, pH is used as a discriminative criterion for the assessment of muscle preparations (Meynial-Denis et al. 1993).

Because the different metabolic ATP supply routes are related to the release or buffering of protons and to lactate production, measurement of pH in muscle could reflect their contribution to the energy homeostasis of muscle. Given the role of glucose as substrate in glycolysis and in oxidative phosphorylation, we thus postulated that the emergence of an acidic component of intracellular pH in muscle might be related to impaired ability of muscle to metabolize exogenous glucose. In this case, intramuscular glycogen stores may be partly catabolized via an anaerobic pathway to provide glucose units available for further degradation.

View larger version (34K): [in this window] [in a new window]   Fig 1. Inorganic phosphate (Pi) and phosphocreatine (PCr) region in spectra of superfused extensor digitorum longus (EDL) muscles from fed and starved rats throughout the experiment: (A) EDL from fed rats and superfusion with glucose, (B) EDL from starved rats and superfusion with glucose, (C) EDL from fed rats and superfusion without glucose, (D) EDL from starved rats and superfusion without glucose. Nuclear magnetic resonance (NMR) acquisition parameters: pulse width 8.5 µs, 120 scans, line-broadening 15 Hz, ± 3250 Hz spectral window and recycle time 5 s. Only a display between 5 and 4 ppm (1462 Hz) is shown. PCr is assigned a chemical shift of 2.45 ppm. Inorganic phosphate (Pi) from extracellular buffer Pi (ex) resonates at 2.8 ppm. Muscle Pi (in) resonates between 1.7 and 2.8 ppm according to experimental conditions. A black arrow is used to indicate Pi peaks identified as split peaks. Each panel (A, B, C or D) is a representative experiment. Each superfusion experiment was repeated 3 to 5 times for each group of rats (fed or starved).

View larger version (13K): [in this window] [in a new window]   Fig 3. Time course of pH changes in extensor digitorum longus (EDL) muscles superfused for 4 h from fed and starved rats. Superfusions of EDL muscle were performed only with 31P-NMR. The intracellular pH component was drawn from the chemical shift of inorganic phosphate (Pi) peak relative to the phosphocreatine (PCr) peak. Standard conditions of superfusion experiments consist in superfusion with Krebs-Henseleit buffer containing glucose (10 mmol/L). pHin, intramuscular pH during the first 30-min stabilization period; pHin1, component1 of intramuscular pH; pHin2, component 2 of intramuscular pH. Values are means ± SEM (n = 5 or 6 experiments).

To determine whether glucose metabolism could influence intramuscular pH measured ex vivo, we studied differences between starved and fed rats. Starved rats are known to display marked changes in skeletal muscle glucose metabolism compared with fed rats. Skeletal muscle from starved rats exhibits higher glucose uptake in vitro (Brady et al. 1981, Goodman and Ruderman 1979, Stirewalt et al. 1985). In vivo, glucose metabolism is markedly slowed due to the decrease in glycemia and impairment of insulin action (Balage et al. 1990, Pénicaud et al. 1985). Intramuscular pH was investigated in isolated and superfused EDL muscles by a direct noninvasive method, ³¹P-nuclear magnetic resonance spectroscopy (³¹P-NMRS). Consequences of the presence or absence of exogenous glucose on muscle pH were analyzed in relation to muscle metabolism.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Muscle superfusion.  Female Wistar rats (Iffa Credo, L'Arbresle, France) were kept in a temperature-controlled room (22 ± 1°C) with a 12-h light:dark cycle. They were divided into two groups of equal mean initial body weight (55 ± 5 g). One group was starved for 48 h but had free access to water; this period of food deprivation induced a 20% loss of initial body weight. The other group was the fed control group, which was given free access to food and water throughout the experimental period. The experiments were conducted in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals. All experiments were performed between 1100 and 1300 h. Rats were anesthetized intraperitoneally (pentobarbital, 45 µg/g body wt), and their EDL muscles dissected out (muscle weight was in the range 20-25 mg irrespective of the nutritional state of the rats). Experiments were performed as previously described (Meynial-Denis et al. 1993) except that superfusion lasted 4 h instead of 2 h. EDL muscles were constantly superfused with Krebs-Henseleit buffer containing bovine serum albumin (1.3 g/L), glucose (10 mmol/L), leucine (3 mmol/L) and 2-oxoglutarate (3 mmol/L). At the end of superfusion, the muscles were immediately frozen and stored in liquid N₂ pending enzymatic assay of ATP, phosphocreatine (PCr), lactate and glycogen. In another set of experiments, fresh samples (EDL muscles and soleus muscles used as controls) and freeze-clamped samples (only EDL muscles) were also taken in situ from anesthetized rats. These last experiments were used to characterize the metabolic properties of muscles and to obtain normal values of ATP, PCr, lactate and glycogen in nonsuperfused muscles.

Biochemical assays.  Lactate, ATP, PCr and glycogen were assayed enzymatically in freeze-dried tissue after muscle deproteinization with perchloric acid (0.4 mol/L) and neutralization with KOH as previously described (Meynial-Denis et al. 1993).

To characterize the EDL muscles by their metabolic properties, freshly excised EDL or soleus muscles from eight fed or eight starved rats were pooled then divided into two equal portions. One part was homogenized in specific extraction buffer (63 mmol/L glycylglycine-NaOH buffer, pH 7.6, containing 500 mmol/L sucrose, 6.2 mmol/L EDTA, 125 mmol/L NaF and 5 mmol/L dithiothreitol) to assay citrate synthase and lactate dehydrogenase (LDH) as reported by Briand et al (1981). From the other part, myofibrillar proteins were isolated in order to measure myofibrillar ATPase activities (see also Briand et al. 1981); the amount of myofibrillar proteins was quantified using the biuret method with bovine serum albumin as standard (Gornall et al. 1949).

³¹P-NMR techniques.  All experiments were performed at 162 MHz in a Bruker AM400 spectrometer (Bruker Spectrospin, Wissembourg, France) using a 10-mm NMR probe. Magnetic field homogeneity was adjusted by observing the proton signal of water (line width ~15 Hz) to obtain a half-width for the phosphocreatine of 5 Hz. Spectra were collected as 8K data points using a sweep width of ±3250 Hz (quadrature detection), a pulse width of 8.5 µs (nominal tip angle 60°) and a recycle time of 5 s. Spectra were the result of 120 transients. No quantification was performed from NMR data. Chemical shifts were expressed with respect to phosphocreatine (which is insensitive to pH variation), which was assigned a chemical shift of 2.45 ppm with respect to 8.67 mmol/L H₃PO₄ (Roberts et al. 1981). The pH was determined from the chemical shift of inorganic phosphate (Pi). A calibration curve of Pi chemical shift against pH was plotted in our experimental conditions (37°C, ionic strength µ = 0.16). From these data, the best fit for pK_a value and chemical shifts of the acid and basic forms of Pi was: pH = 6.824 + log [() 0.922)/(3.451  )] where  is the chemical shift of Pi. The curve fitting and calculation were done with a nonlinear regression program (SAS statistical package, Cary, NC). Because the Pi chemical shift is a very sensitive probe of pH discrete compartmentation, it was used to estimate both extracellular pH (from superfusion buffer) and intramuscular pH (Meynial-Denis et al. 1993). To obtain optimal accuracy in the pH measurements and hence reliable evidence for any compartmentation within the muscle, NMR data were processed by the curve-fitting treatment with an inverse-polynomial function (NMR_i, NMRi, Syracuse, NY).

Statistical analysis.  Each superfusion experiment (in situ or superfused) was repeated three to five times for each group of rats (fed or starved). It should be pointed out that, in each experiment, it was necessary to use EDL muscles from four different rats in NMR superfusion experiments. Values are means ± SEM. Two-way ANOVA was used to discriminate the effects among superfusion, nutritional state and their interactions. Differences are considered significant at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

pH and energy metabolism of 4-h superfused EDL muscles with glucose.  Because there were no differences in ATP and PCr concentrations in relation to the nutritional state of rats (Table 1), only the region of inorganic phosphate (Pi) was studied in ³¹P-NMR spectra of superfused EDL muscles. This region of the ³¹P spectra was very different between fed and starved animals during glucose superfusion (Fig. 1A and 1B). Because the intramuscular Pi component was clearly distinguishable from extracellular Pi and the pH calculation was based on chemical shift of Pi peak, both the superfusion buffer pH (which remained constant and equal to 7.3-7.4 during the experiment) and the intramuscular pH (which can change within the time and the conditions of superfusion) were measured. The following should be noted for the first 30 min of superfusion: 1) a single pH value appeared as calculated in Fig. 2A and 2) a significant difference was observed in the intracellular Pi peak between starved and fed rats. pH was 6.94 ± 0.03 (n = 5) and 6.71 ± 0.04 (n = 6), P < 0.05) in starved and fed rats, respectively. The intracellular pH in EDL muscles from starved rats continued to differ from that of fed rats throughout the course of glucose superfusion (illustrated in Figs. 1A and 1B). Comparison of the time course of intramuscular pHs between fed and starved rats is shown in the Fig. 3A and B, respectively. In fed rats, the intracellular Pi peak was split into two portions, i.e., the pH exhibited two components: pH_in1 in the range 6.8 to 7 and pH_in2 in the range 6.6 to 6.75 (analyzed as reported in Fig. 2.C). Component pH_in1 was the main component of the intramuscular pH. In contrast, in muscles from starved rats, intracellular pH consisted of a single component (pH_in1), which was higher than in fed rats during the experiment. Its value (within 7-7.3) could not be determined accurately because the intramuscular Pi peak overlapped with the extracellular Pi peak (see analysis in Fig. 2B).

View larger version (17K): [in this window] [in a new window]   Fig 2. Curve-fitting treatment of nuclear magnetic resonance (NMR) spectra to determine accurate values of muscle pH: (A) Inorganic phosphate define (Pi) (in) appears as a distinct peak of Pi (ex) (Example of the spectrum 16 min in Fig. 1D). (B) Pi (in) overlaps Pi (ex) (Example of the spectrum 186 min in Fig. 1B). (C) Pi (in) appears as a distinct peak of Pi (ex) and gives rise to a split peak (Example of the spectrum 106 min in Fig. 1D). In each panel, four spectra are shown: x, experimental spectrum; m, calculated spectrum; a, fitted peaks and d, difference spectrum).

Analysis of lactate and glycogen levels enabled us to demonstrate other differences between fed and starved rats. The lactate level was found to be slightly higher (P < 0.05) in fed than in starved rats in situ or in EDL muscles superfused for 4 h (Table 1). In contrast, the significant difference shown in in situ muscles from fed and starved rats disappeared during the 4-h superfusion with glucose. Indeed, the glycogen level was similar in EDL muscles superfused for 4 h from fed and starved rats. In other words, glycogen depletion induced by food deprivation was restored by glucose supply. No effect of glucose superfusion was observed on lactate level regardless of the nutritional state of animals (see Table 1).

Effect of glucose deprivation during superfusion.  The metabolism of ex vivo EDL muscles closely depends on exogenous substrates for oxidative metabolism. Therefore, to assess the importance of glucose supply, we tested the muscle bioenergetics in EDL muscles superfused for 4 h with buffer without the inclusion of glucose. Typical ³¹P-NMR spectra are displayed in Figs. 1C and 1D. After the first 30 min, the muscle Pi peak split, as previously observed in fed muscle, into two components of pH (pH_in1 and pH_in2), which were maintained until the end of the 4-h superfusion, irrespective of the nutritional state of the rats (Figs. 4A and 4B). Analysis of these pH components was conducted after curve fitting as reported in Figs. 2A and 2C. We also demonstrated that the glycogen concentration could not be restored in EDL muscle from starved rats as it was when glucose was present (Table 2). The absence of glucose did not alter the glycogen concentration in muscle from fed rats. Glucose deprivation depressed the lactate level to the same extent in both groups of rats (fed and starved), i.e., to ~20% of the value with glucose (see Table 2).

View larger version (31K): [in this window] [in a new window]   Fig 4. Effect of glucose supply to superfused extensor digitorum longus (EDL) muscles on time course of changes in intramuscular pH in fed and starved rats. The perfusate was Krebs-Henseleit buffer without glucose; additional substrate [glucose or 2-deoxyglucose (2-DOG)] is indicated in each panel when necessary. In panels A and B, glucose was absent for the whole period. In panels C and D, glucose was present for only the last 2 h. In panels E and F, glucose was replaced by 2-DOG. See legend of Figure 3 for abbreviations. Values are means ± SEM (n = 3-4 experiments).

To analyze the reversibility of the changes introduced by the absence of glucose, muscles underwent a 2-h period without glucose followed by 2 h with glucose replacement (referred to as period 1 and period 2, respectively, in Table 2). After glucose addition, this protocol also yielded one pH component in muscle from starved rats. However, this pH value remained lower than that in an experiment in which glucose was always present (~7 vs. a value in the range of 7 to 7.3; see Fig. 4D). Glucose addition blunted the alterations in lactate levels in both groups (Table 2). In contrast, glucose was unable to restore glycogen level in starved animals (Table 2).

To emphasize the role of glucose in cell energy homeostasis, we performed 4-h experiments in which glucose was omitted during the first 2 h and replaced during the last 2 h by the nonmetabolizable analog of glucose, 2-deoxy glucose (2-DOG) (see Table 2). As expected, the glycogen content was not restored in starved rats using 2-DOG, and the pH exhibited two components during the 4 h in starved rats (Fig. 4F) instead of one component when glucose was added after a period of glucose deprivation (Fig. 4D). Low levels of lactate (Table 2) in these experiments reflected the fall in glucose metabolism through the glycolytic pathway.

Other metabolic characteristics of muscle.  To explain differences in glucose metabolism between EDL muscles from fed and starved rats, we further characterized their metabolic type (oxidative or glycolytic). The soleus muscle, which is known as a highly oxidative muscle (Smith et al. 1988) was used as control. Metabolic characteristics of muscles were assessed by enzyme activity measurements (citrate synthase, lactate dehydrogenase or LDH, ATPase). As expected, in the fed state, ATPase and LDH activities were higher in EDL muscle [0.75 ± 0.08 µeq/(min·mg protein) and 6.98 ± 1.00 units/g wet tissue, respectively] than in soleus muscle [0.44 ± 0.05 µeq/(min·mg protein) and 2.31 ± 0.24 units/g wet tissue, respectively], whereas the citrate synthase activity was similar (about 0.45 units/g wet tissue). In EDL muscles, 48-h food deprivation induced an increase in the citrate synthase activity (0.70 ± 0.01 units/g wet tissue, P < 0.05 vs. fed rats) but no change in ATPase activity. Consequently, starvation induced significant increases in the oxidative character of EDL muscle. In contrast, this treatment did not induce any modification in the metabolic properties of the soleus muscle.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The major finding in this study is that the intramuscular pH, measured by ³¹P-NMR, is a discriminative criterion between ex vivo resting EDL muscle preparations from starved and fed rats. This is the first study, to our knowledge, providing evidence that the intramuscular pH measured ex vivo depends on the prior nutritional state of animals. This difference was not detectable in resting muscle of rats studied in vivo (Jacobs et al. 1985, Pichard et al. 1988). This last point underlines the advantage of using ex vivo vs. in vivo experiments to assess muscle metabolism in the presence of an exogenous substrate such as glucose. Indeed, the pH value, which was always higher in glucose-superfused muscles of starved than fed rats, may reflect a metabolic adaptation in skeletal muscle to food deprivation. Lactate levels in these muscles (twofold higher in fed than in starved rats, even before the superfusion experiment) support this hypothesis. In muscles from fed rats, the main component of intramuscular pH (pH_in1) remained close to a neutral value and did not reach a plateau at a slightly alkaline value (between 7 and 7.3) as in starved rats. This could result from glucose being routed towards anaerobic glycolysis rather than oxidative phosphorylation. The higher activity of citrate synthase in starved than fed rats in consistent with this explanation. These differences in metabolic capacity are not related to the profile of the fiber type determined histochemically [about 60% fast-twitch oxidative glycolytic fibers, 30% fast-twitch glycolytic fibers and about 10% slow-twitch oxidative fibers irrespective of the nutritional state of animals (Meynial-Denis et al. 1993 and unpublished data). This is evidence that to elucidate the true metabolic character of a muscle, measurements of enzyme activities within the muscles and not just data concerning fiber type are required (Baillie and Garlick 1991). Differences between fed and starved rats may be related to the ability of fast-twitch oxidative glycolytic fibers to alter their metabolism (Pette and Staron 1990). Consequently, in starved rats, oxidative phosphorylation may be strongly stimulated to produce large amounts of ATP without acidification of muscle and help maintain a high value of intramuscular pH_in1. An analogy with processes observed in EDL muscles in vivo during fasting/refeeding, leading to an increase of glucose oxidation (Holness et al. 1988 and 1989, Holness and Sugden 1991, Issad et al. 1987, Sugden et al. 1989, Sugden and Holness 1989), seems to be consistent with this analysis.

Another major finding in this work is that a second pH component (pH_in2), more acidic than pH_in1, is always observed in EDL muscle from fed rats. The pH splitting in muscles of fed rats could not arise from compartmentation of fibers, i.e., separate glycolytic and oxidative fiber compartments each giving a discrete pH value. The fiber type composition in muscle from fed rats is not different than that in starved rats (see below). Moreover, the small component pH_in2 appears in EDL muscles of starved rats only if glucose is absent or replaced by a nonmetabolizable analog of glucose in the medium superfusion. Consequently, this might result from very small regions of anaerobic glycogenolysis to maintain energy homeostasis even though muscle size is very small. It should be pointed out that this local decrease in glycogen (coupled with a local lactate increase) was minor and not large enough to give rise to detectable changes in glycogen and lactate concentrations in the whole muscle. Unfortunately, this study did not provide a direct line of evidence with which to determine the validity of this speculation. Further studies are required to conclusively elucidate the mechanism (for example, sophisticated experiments of lactate imaging within the muscle).

In EDL muscle from fed rats, glycogen remained at basal levels and the intracellular pH always exhibited the two components, irrespective of the conditions of superfusion. In other words, the intracellular pH components in muscle of fed rats were not sensitive to the presence or absence of glucose. The alteration of the utilization of glucose in fed rats could be explained on the basis of glucose transporter Glut1. Both the protein and the mRNA levels of this transporter were lower in skeletal muscle from fed than in starved mammals (Burant et al. 1991, Wertheimer et al. 1991). Glucose transport through Glut1 could thus be the rate-limiting step in glucose utilization by the muscle in the absence of insulin.

Exogenous glucose also plays a major role in glycogen repletion in EDL muscles from starved rats. It has been shown (Holness et al. 1988, Holness and Sugden 1991, Sugden et al. 1990), and was confirmed by our results that food deprivation leads to glycogen store depletion (~50% after 2 d of food deprivation). Consequently, glucose utilization through the glycogen synthesis pathway may be more important than the oxidative pathway. Indeed, glucose superfusion for 4 h completely restored the glycogen in EDL muscles from starved rats. This has also been reported in various skeletal muscles studied for shorter times (Brady et al. 1981, Goodman et al. 1974, Goodman and Ruderman 1979, Stirewalt et al. 1985). Rapid total replenishment of muscle glycogen has also been observed in vivo after refeeding as a result of high rates of glycogen deposition. There was a clear inverse relationship between initial rate of glycogen deposition and extent of glycogen depletion during food deprivation (Holness et al. 1988).

In conclusion, we demonstrate from the ³¹P-NMR chemical shift of Pi that the intramuscular pH measured ex vivo in the presence of glucose depends on the nutritional state of rats. The pH always higher in glucose-superfused muscles of starved than fed rats, may reflect a metabolic adaptation in skeletal muscle to rats food deprivation. The appearance of a second and lower pH compartment (always observed in the case of EDL muscles from fed rats and only in muscle of starved rats if glucose was removed from the medium) suggests that impairment of glucose metabolism inducing local glycogenolysis can generate acidosis and pH compartmentation in skeletal muscle. Consequently, direct pH measurement by ³¹P-NMR may be considered a precise criterion for evaluating differences in metabolic properties of muscle ex vivo.

	ACKNOWLEDGMENTS

The authors thank Didier Attaix, Philippe Patureau-Mirand, Thomas Vary and Susan Samuels for their critical review of the manuscript, Danielle Bonin and Helene Lafarge for contributions to the bibliography and Rachel Tardy for her help in the preparation of the manuscript.

	FOOTNOTES

Manuscript received 25 October 1996. Initial reviews completed 28 February 1997. Revision accepted 3 September 1997.

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