磷酸肌酸和肌酸对骨骼肌细胞线粒体呼吸的影响

The control of mitochondrial respiration is a cardinal issue in the field of muscle energetics. Early work on isolated mitochondria identified ADP as an important stimulator of mitochondrial respiration (Lardy & Wellman, 1952; Chance & Williams, 1955). Later, the [ADP]/[ATP] ratio or the inverse phosphorylation potential [ADP] [Pi]/ [ATP] as well as the redox state of the electron transport chain were recognised as important regulators of respiration (for a review see Balaban, 1990). Although isolated mitochondria have proved to be an ideal tool for measurements of certain parameters of mitochondrial control and function (e.g. coupling efficiency), some of the sophisticated respiratory control mechanisms present in intact skeletal muscle are lost during the isolation procedure. For example the sensitivity of respiration to ADP and the effect of creatine (Cr) appear to be altered during the isolation procedure (Saks et al. 1995). An additional experimental limitation is that only a fraction (10–25 %) of the mitochondria are harvested from the muscle. An alternative method for studying muscle oxidative function is the use of chemically permeabilised (skinned) muscle fibres. Unlike isolated mitochondria, skinned fibres allow virtually the entire mitochondrial population of the muscle sample to be studied in their natural The role of phosphorylcreatine and creatine in the regulation of mitochondrial respiration in human skeletal muscle B. Walsh *, M. Tonkonogi *†, K. Söderlund *†, E. Hultman ‡, V. Saks § and K. Sahlin *† *Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden, † Stockholm University College of Sport Science, Stockholm, Sweden, ‡Department of Clinical Chemistry, Huddinge University Hospital, Karolinska Institute, Huddinge, Sweden and § Institute of Chemical Physics and Biophysics, Tallinn, Estonia (Received 15 June 2001; accepted after revision 4 September 2001) 1. The role of phosphorylcreatine (PCr) and creatine (Cr) in the regulation of mitochondrial respiration was investigated in permeabilised fibre bundles prepared from human vastus lateralis muscle. 2. Fibre respiration was measured in the absence of ADP (V0) and after sequential additions of submaximal ADP (0.1 mM ADP, Vsubmax), PCr (or Cr) and saturating [ADP] (Vmax). 3. Vsubmax increased by 55 % after addition of saturating creatine (P < 0.01; n = 8) and half the maximal effect was obtained at 5 mM [Cr]. In contrast, Vsubmax decreased by 54 % after addition of saturating phosphorylcreatine (P < 0.01; n = 8) and half the maximal effect was obtained at 1 mM [PCr]. Vmax was not affected by Cr or PCr. 4. Vsubmax was similar when PCr and Cr were added simultaneously at concentrations similar to those in muscle at rest (PCr/Cr = 2) and at low-intensity exercise (PCr/Cr = 0.5). At conditions mimicking high-intensity exercise (PCr/Cr = 0.1), Vsubmax increased to 60 % of Vmax (P < 0.01 vs. rest and low-intensity exercise). 5. Eight of the subjects participated in a 16 day Cr supplementation programme. Following Cr supplementation, V0 decreased by 17 % (P < 0.01 vs. prior to Cr supplementation), whereas ADP-stimulated respiration (with and without Cr or PCr) was unchanged. 6. For the first time evidence is given that PCr is an important regulator of mitochondrial ADP- stimulated respiration. Phosphorylcreatine decreases the sensitivity of mitochondrial respiration to ADP whereas Cr has the opposite effect. During transition from rest to high- intensity exercise, decreases in the PCr/Cr ratio will effectively increase the sensitivity of mitochondrial respiration to ADP. The decrease in V0 after Cr supplementation indicates that intrinsic changes in membrane proton conductance occur. Journal of Physiology (2001), 537.3, pp.971–97812858 971 Administrator Highlight Administrator Sticky Note Administrator Callout 股外侧肌 structural environment (i.e. the connections between the mitochondria and the cytoskeleton remain undisturbed) (Kay et al. 1997; Saks et al. 1998). All the soluble cytosolic enzymes and metabolites are removed during the preparation, enabling experimental manipulations of the environment surrounding the functionally intact mitochondria (Kay et al. 1997; Saks et al. 1998). In contrast to isolated mitochondria, the connections between mitochondria and cytoskeleton are preserved in skinned fibres and this is considered to be crucial for the maintenance of a high Km for ADP and for the ability of Cr to stimulate respiration (Saks et al. 1991, 1994, 1995, 1998; Kay et al. 2000). Creatine kinase (CK) is located in the cytosol and in the mitochondrial intermembrane space (CKmit). The location of CK in the muscle cell, together with the low permeability of the outer mitochondrial membrane for adenine nucleotides, is the basis for the Cr shuttle model of muscle energetics (Saks et al. 1976; Bessman, 1985). The model states that Cr is transported from the ATP utilising sites (e.g. myofibrils) to mitochondria, while PCr is transported in the reverse direction. Due to the presence of CKmit at the inner mitochondrial membrane, Cr will react with ATP formed by oxidative phosphorylation. This will increase local [ADP] and stimulate respiration. There is evidence that Cr-stimulated respiration is an important feature of oxidative muscles, including cardiac tissue, but is absent in fast-twitch muscles (Kuznetsov et al. 1996). Recent data demonstrate that mitochondria are incorporated into functional complexes with the ADP-producing systems (Seppet et al. 2001). Since the CK reaction is reversible, increased [PCr] is expected to decrease [ADP] and thus have the opposite effect to creatine on mitochondrial respiration. Therefore, we hypothesise that the presence of PCr may reduce respiration at rest and that decreases in the PCr/Cr ratio during exercise may be an important activator of respiration in vivo. If both PCr and Cr modulate mitochondrial respiration, it is of physiological importance to investigate the sensitivity of respiration to changes in [Cr] and [PCr] and combinations of PCr and Cr that mimic in vivo conditions. Dietary supplementation with Cr has been shown to increase intramuscular levels of PCr + Cr (TCr) (Harris et al. 1992) and has been shown to enhance performance during high-intensity exercise (Balsom et al. 1993; Greenhaff et al. 1993). It is well known that the CK reaction is the most rapid process for ATP generation and increased PCr will therefore effectively increase the energetic power and may, at least partly, explain the improvement in supramaximal performance after Cr supplementation. The influence of Cr supplementation on oxidative energy supply has not been studied in detail. Since endurance during moderate intensity exercise is not improved by Cr supplementation (Balsom et al. 1993; Stroud et al. 1994; Vandebuerie et al. 1998), it may be argued that oxidative function during the steady state is not affected by increased [Cr]. However, it is possible that, at least in some of these studies, the increased body weight associated with Cr supplementation has confounded the results (Balsom et al. 1993; Stroud et al. 1994). There is some evidence to support the hypothesis that creatine supplementation enhances oxidative phosphorylation. First, it has been shown that Cr supplementation can increase the rate of PCr resynthesis during recovery after intense exercise (Greenhaff et al. 1994). Second, during low-intensity exercise performed intermittently, utilisation of PCr was reduced after Cr supplementation (Rico-Sanz, 2000). These findings may be due to an effect of increased [Cr] as such but may also be due to intrinsic changes in mitochondrial function. The effect(s) (if any) of Cr supplementation on oxidative function remains elusive and measurements with the skinned fibre technique may provide a mechanistic approach to the problem. The purpose of this study is (i) to investigate the physiological role of PCr in oxidative metabolism, (ii) to assess the sensitivity of respiration to Cr and PCr and the effects of physiological combinations of [PCr] and [Cr] on respiration, and (iii) to investigate whether Cr supplementation alters mitochondrial respiratory control. METHODS Subject data Thirteen healthy male subjects participated in the study. The subjects’ mean age, weight and height (range) were: 24.8 (19–28) years, 81.3 (73.5–96.0) kg and 179.9 (173.0–189.5) cm, respectively. None of the subjects who participated in the study had used Cr as a dietary supplement but no measurements or tests were conducted to verify this statement. All subjects were fully informed of the possible risks and discomforts involved in the experiment before giving their written voluntary consent. The experimental design of the study was approved by the Ethics Committee of the Karolinska Institute, Stockholm, Sweden. All experiments conformed with the Declaration of Helsinki. Experimental procedure Maximal oxygen uptake during cycling (VO2,peak) was determined on a Monark 829e cycle. Subjects performed a discontinuous incremental cycle ergometer test (80 r.p.m.) until exhaustion. Expired air was collected in Douglas bags and analysed for O2 and CO2 (Beckman Instruments, Fullerton, CA, USA). Two to three days following the VO2,peak test, muscle biopsies were taken from the vastus lateralis. After local anaesthesia (1–2 ml carbocain; 20 mg ml_1, Astra), an incision was made through the skin and fascia at one-third of the distance from the upper margin of patella to the anterior superior iliac spine. The biopsies were performed using the technique of Bergström with suction, and were randomised between legs. Each biopsy was divided with a surgical blade into at least two portions. One portion was frozen and stored at _80 °C until analysis of fibre type composition. A second portion was further separated into several small sections and placed in an ice-cold medium consisting of (mmol l_1): EGTA–CaEGTA buffer, 10 (free Ca2+ concentration 100 nM); imidazole, 20; KH2PO4, 3; dithiothreitol, 0.5; ATP, 5.3; PCr, 15; MgCl2, 9.5; 2-[N-morpholino]ethanesulphonic acid B. Walsh and others972 J. Physiol. 537.3 (Mes), 53.5; pH 7.00. The fibre bundles were separated with sharp- ended needles, leaving only small areas of contact, and incubated in 1.5 ml of the above medium (4 °C) containing 50 µg ml_1 saponin for 30 min with mild stirring. In order to completely remove saponin and metabolites, the fibres were washed three times with mild stirring for 5 min in 1.2 ml of cooled (4 °C) washing and oxygraph medium consisting of (mmol l_1): EGTA–CaEGTA buffer, 10 (free Ca2+ concentration 100 nM); imidazole, 20; KH2PO4, 3; pyruvate, 5; malate, 2; MgCl2, 4; Mes, 100; dithiothreitol, 0.5; taurine 20; and BSA, 2 mg ml_1; pH 7.00. After washing, the fibres were stored on ice until determination of respiratory activity. The maximal time between fibre preparation and measurement of respiratory activity was 2.5 h. Previous studies in our laboratory have shown that fibre respiration remains unchanged during this period (Tonkonogi et al. 1998). In eight subjects, a third portion was immediately quenched in liquid nitrogen and stored at _80 °C for metabolite determination. Measurements of mitochondrial respiration Mitochondrial oxygen consumption was measured polargraphically with a Clark-type electrode (Hansatech DW1, King’s Lynn, Norfolk, UK) in a water-jacketed glass chamber maintained at 25 °C. Measurements were carried out in 0.3 ml of the above-described washing and oxygraph solution. In all fibres, respiration was measured in the absence of ADP (V0) and following the addition of 0.1 mM ADP (Vsubmax). Following measurement of V0 and Vsubmax, respiration in skinned fibres from eight subjects was measured after sequential additions of Cr (final [Cr] 5 mM and 20 mM) and ADP (Vmax, final [ADP] 5 mM). In another fibre bundle (from the same muscle biopsy) respiration was measured after sequential additions of PCr (final [PCr] 1 mM and 20 mM) and ADP (Vmax, final [ADP] 5 and 10 mM). Due to the inhibition of respiration with PCr a higher concentration of ADP was required to reach Vmax. The effect of different combinations of PCr + Cr was investigated in muscle samples from five subjects. Following measurement of respiration at Vsubmax, PCr and Cr were added at concentrations (mM) similar to those in muscle at rest (24 PCr + 12 Cr; PCr/Cr = 2), and during low-intensity work (12 PCr + 24 Cr; PCr/Cr = 0.5) or high-intensity work (3 PCr + 33 Cr; PCr/Cr = 0.1). Mitochondrial respiration was maximally stimulated by the addition of ADP to a final concentration of 10 mM (Vmax). Immediately following respiratory measurements, the fibre bundles were removed, quick frozen in liquid nitrogen, freeze-dried and weighed. Wet weight was used as a reference base for respiration and was obtained from the dry weight assuming 77 % water content (Bergström, 1962). Creatine supplementation After the first biopsy, the eight subjects in whom the sensitivity of mitochondrial respiration to PCr or Cr was studied began a 16 day creatine supplementation programme. During the first 5 days of supplementation 20 g creatine + 180 g glucose were ingested daily (divided into two equal portions taken in the morning and evening). During days 6–16 of the supplementation period subjects ingested 2 g creatine + 18 g glucose (one portion per day). On day 17, muscle biopsies were taken, and fibre respiration was measured with an identical procedure to the first muscle biopsy. Fibre type determination Part of each biopsy was used for fibre type determination. Muscle samples were mounted in an embedding medium (Tissue-Tek O.C.T. Compound 4583, Histolab Products AB, Frolunda, Sweden), frozen in liquid nitrogen-cooled isopentane and stored at _80 °C. Transverse sections (6 µm) were cut with a cryostat (Leica Jung Frigocut 2800E) and the slides were stored at _80 °C. The fibre sections were incubated in 2 % formaldehyde solution for 20 min, rinsed in distilled water, and washed in Earle’s balanced salt solution (EBSS) (14050–041; Life Technologies, Paisley, UK) (including Hepes 1 % and saponin 0.1 %) for 3 w 3 min. The fibres were prepared for the addition of the primary antibody through incubation in a solution containing EBSS + saponin + 1 % H2O2 + 2 % NaN3 in darkness for 1 h. The fibres were then rinsed for 3 w 3 min in EBSS and the primary antibody (monoclonal anti-myosin (skeletal, slow) M8421, Sigma) was added. Incubation with the primary antibody lasted 18 h. After overnight storage, the fibres were washed in EBSS for 3 w 3 min and exposed to Histostain Plus reagents A, B and C (commercial kit available from Zymed Laboratories Inc., San Francisco, USA). Following treatment with Histostain Plus, the slides were developed in an AEC (3-amino-9-ethyl-carbazole, A-6926, Sigma) solution (30 mg AEC + 12 ml DMSO + 0.002 % H2O2/100 ml EBSS buffer) for 10 min in darkness. The slides were washed for 3 w 3 min and the background was coloured by exposing the slides to Mayer’s haematoxylin solution (Apoteket, Sweden) for 30–40 s. In six of the eight subjects participating in the Cr supplementation programme it was possible to determine fibre type composition in both pre- and post-supplementation samples. There was no effect of Cr supplementation on the relative numbers of type I fibres or fibre type area following the supplementation period (n = 6; data not shown). Metabolite analysis PCr and Cr were analysed in the biopsy samples from the subjects who participated in the Cr supplementation programme. The samples were freeze-dried and dissected free of solid non-muscle constituents, powdered, and extracted with perchloric acid (0.5 mol l_1) and neutralised with KHCO3 (2.2 mol l _1). Analyses were performed by NAD(P)H-coupled specific enzymatic reaction adapted for spectrophotometric assays of NAD(P)H (Harris et al. 1974). Data analysis All values are presented as means ± S.E.M. Differences between means were tested for statistical significance with Student’s paired t test. Significance was set at P < 0.05. RESULTS The subjects’ maximal oxygen uptake reached during the cycle test (VO2,peak) was 4.2 ± 0.1 l min _1 or 52.2 ± 1.9 ml O2 (kg body weight) _1 and the area of type I fibres in the vastus lateris was 53.1 ± 3.9 % (n = 13). Representative oxygraphic traces of mitochondrial respiration in permeabilised muscle fibres after sequential additions of submaximal ADP, PCr (or Cr), and saturating ADP concentrations are shown in Fig. 1. Muscle fibre respiration in the presence of 0.1 mM ADP (Vsubmax) averaged 42 % of maximal ADP-stimulated respiration (Vmax, n = 13). Addition of 20 mM PCr (sufficient to elicit maximal effect; data not shown) decreased respiration by 54 % (P < 0.01, n = 8, Fig. 2A). About half the maximal effect of PCr was achieved at 1 mM. In contrast, the addition of 20 mM Cr (sufficient to elicit maximal effect; data not shown) to skinned muscle fibres respiring at 0.1 mM ADP increased respiration by 55 % (P < 0.01, n = 8, Fig. 2B). The addition of 5 mM Cr was sufficient to reach half the maximal Cr-stimulated increase in respiration. Although the presence of PCr or Cr decreased or increased the respiration in the presence of 0.1 mM ADP, Vmax was identical regardless of the presence of PCr or Cr Control of fibre respiration by PCr and CrJ. Physiol. 537.3 973 (1.87 ± 0.12 vs. 1.85 ± 0.12 mmol O2 min _1 (kg wet wt)_1, respectively). Therefore, additions of PCr or Cr altered the sensitivity of respiration to ADP ((Vsubmax _ V0)/(Vmax _ V0)). In the presence of 20 mM PCr the ADP sensitivity decreased by 84 ± 4 %, while 20 mM Cr increased the ADP sensitivity by 83 ± 10 %. The effect of three combinations of PCr + Cr on fibre respiration at a constant [ADP] (0.1 mM) was studied. The combinations of Cr + PCr were chosen to mimic in vivo concentrations (mM) at rest (24 PCr/12 Cr; PCr/Cr = 2), during low-intensity exercise (LI: 12 PCr/24 Cr; PCr/Cr = 0.5) and during high-intensity exercise (HI: 3 PCr/33 Cr, PCr/Cr = 0.1). Respiration at LI conditions was similar to that at rest, whereas under HI conditions it was about twofold higher (P < 0.01 vs. both rest and low- intensity work conditions; Fig. 3). Eight subjects participated in a creatine supplementation programme for 16 days. TCr was higher in seven of the eight subjects after the supplementation period but due to a decrease in one subject the difference did not reach statistical significance (Table 1). This subject had already an exceptionally high TCr prior to the supplementation period (146.4 mmol (kg dry wt)_1). V0 decreased by 17 % (P < 0.01) following Cr supplementation; however, neither Vsubmax, Vmax nor the rate of respiration in the presence of PCr or Cr were altered following the supplementation period (Table 1). DISCUSSION The primary finding of this study was that PCr reduces submaximal respiration but not maximal ADP-stimulated respiration. Previously, several investigators have reported that respiration increases when Cr is added to the respiration medium (Kuznetsov et al. 1996; Tonkonogi et al. 1998, 1999). The effect is believed to be due to the Cr shuttle system within skeletal muscle by which Cr will increase the local concentration of free ADP (see Introduction). Increases in [ADP] in the mitochondrial intermembrane space will increase respiration since ADP is a potent stimulator of respiration. The finding that PCr reduces respiration is fully compatible with the Cr shuttle hypothesis. The CK reaction is reversible and, therefore, the presence of PCr will reduce [ADP] and mitochondrial respiration. The maximal effect of PCr and Cr was reached at 20 mM and the magnitude (expressed either as change in ADP sensitivity or the relative change in Vsubmax) was nearly identical. However, the concentration required to reach half the maximal effect was lower for PCr (1 mM)