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• W1598394864 abstract "Creatine (Cr) plays an important role in muscle energy homeostasis as a substrate in the creatine kinase phosphoryl exchange reaction, but the consequences of creatine depletion are incompletely understood. We assessed the morphological, metabolic and functional consequences of systemic creatine depletion on skeletal muscle in a mouse model with deficiency of an essential enzyme in the biosynthesis of creatine (AGAT−/− mice). We show that Cr depletion leads to several metabolic abnormalities in muscle, including reduced ATP, increased inorganic phosphate levels and reduced activities of proton-pumping respiratory chain enzymes and an elevated glycolytic contribution in ischaemic circumstances. The Cr-depleted muscle suffers from reduced grip strength, severe atrophy and abnormal mitochondrial structures, increased overall mitochondrial content and an increased number of lipid droplets. Oral Cr administration led to rapid accumulation in skeletal muscle (faster than in brain) and reversed all the muscle abnormalities, revealing that the condition of the AGAT−/− mice can be switched between Cr deficient and normal simply by dietary manipulation. Abstract Creatine (Cr) plays an important role in muscle energy homeostasis by its participation in the ATP–phosphocreatine phosphoryl exchange reaction mediated by creatine kinase. Given that the consequences of Cr depletion are incompletely understood, we assessed the morphological, metabolic and functional consequences of systemic depletion on skeletal muscle in a mouse model with deficiency of l-arginine:glycine amidinotransferase (AGAT−/−), which catalyses the first step of Cr biosynthesis. In vivo magnetic resonance spectroscopy showed a near-complete absence of Cr and phosphocreatine in resting hindlimb muscle of AGAT−/− mice. Compared with wild-type, the inorganic phosphate/β-ATP ratio was increased fourfold, while ATP levels were reduced by nearly half. Activities of proton-pumping respiratory chain enzymes were reduced, whereas F1F0-ATPase activity and overall mitochondrial content were increased. The Cr-deficient AGAT−/− mice had a reduced grip strength and suffered from severe muscle atrophy. Electron microscopy revealed increased amounts of intramyocellular lipid droplets and crystal formation within mitochondria of AGAT−/− muscle fibres. Ischaemia resulted in exacerbation of the decrease of pH and increased glycolytic ATP synthesis. Oral Cr administration led to rapid accumulation in skeletal muscle (faster than in brain) and reversed all the muscle abnormalities, revealing that the condition of the AGAT−/− mice can be switched between Cr deficient and normal simply by dietary manipulation. Systemic creatine depletion results in mitochondrial dysfunction and intracellular energy deficiency, as well as structural and physiological abnormalities. The consequences of AGAT deficiency are more pronounced than those of muscle-specific creatine kinase deficiency, which suggests a multifaceted involvement of creatine in muscle energy homeostasis in addition to its role in the phosphocreatine–creatine kinase system. Phosphocreatine (PCr) is a major source of ATP replenishment in tissues with rapidly fluctuating energy demand. This supply is mediated by the creatine kinase (CK) reaction, in which creatine (Cr) and ADP are reversibly phosphorylated to PCr and ATP, respectively. The PCr–CK system, which functions as a spatial and temporal buffer of ATP levels, requires a high level of total cellular Cr; 20–40 mm in mammal skeletal muscle (Wyss & Kaddurah-Daouk, 2000). High intracellular Cr concentrations are accomplished by a combination of endogenous production and exogenous dietary intake, followed by cellular uptake of Cr from blood vessels (Fig. 1). Biosynthesis of creatine De novo synthesis of creatine mainly takes place in the kidneys, pancreas and liver. The first step of the biosynthesis of creatine (Cr) is rate limiting and is catalysed by l-arginine:glycine amidinotransferase (AGAT). The second step is catalysed by guanidinoacetate methyltransferase (GAMT). The produced Cr is transported by Cr transporters (CRT) towards tissues that have a high energy demand, such as muscle or brain, where it is phosphorylated in the creatine kinase (CK) reaction, which plays an important role in maintaining ATP levels. A proportion (∼1.5%) of the total Cr is converted non-enzymatically into creatinine (Crn), which is excreted by the kidneys. The PCr–CK system of cardiac and skeletal muscle has been thoroughly investigated in transgenic mouse models with partial or complete deletions or overexpression of muscle-specific CK isoforms. These studies have been valuable in defining the role and importance of the PCr–CK system in skeletal muscle in different circumstances, e.g. at rest, upon stimulation or during ischaemia (for reviews see Nicolay et al. 1998; Heerschap et al. 2007; Saks et al. 2007; Salomons & Wyss, 2007; Saks, 2008). Surprisingly, skeletal muscle of double knockout mice with complete absence of both the cytosolic (M-CK) and mitochondrial (Sc-CKmit) CK isoforms still contains a substantial amount of PCr; however, this cannot easily be recruited for ATP buffering, and the consequent lack of burst activity shows the particular importance of the ATP-buffering role of the PCr–CK system during the initial phase of intense muscle contraction (de Haan et al. 1995; Steeghs et al. 1998). As these double knockout mice still contain substantial amounts of (P)Cr, they cannot be used as a model to study the ultimate consequence of Cr absence. Previous studies have explored the administration of Cr analogues to replace creatine, in particular β-guanidinopropionic acid (β-GPA). This has several effects on muscular phenotype and biochemistry, which include reduced ATP levels, a switch from fast to slow myosin isoform expression (Moerland et al. 1989) and decreased muscle fibre diameters (Shoubridge et al. 1985). However, the supplemented β-GPA can still be phosphorylated and used as a high-energy phosphate (HEP) analogue and thus compensate at least partly for the absence of PCr in skeletal muscle. To study the effect of pure Cr deficiency in muscle, therefore, requires disruption of Cr biosynthesis. Severely depleted Cr levels have been reported in patients with defects in the expression of one of the two enzymes of de novo Cr synthesis, namely l-arginine:glycine amidinotransferase (AGAT; EC 2.1.4.1) and guanidino acetate methyltransferase (GAMT; EC 2.1.1.2). Although GAMT−/− mice are essentially free of (P)Cr (Schmidt et al. 2004), they accumulate guanidino acetate, one of the precursors of Cr, as well as its phosphorylated form, PGAA (Renema et al. 2003). As PGAA is able to buffer ATP in (mildly) energy-demanding conditions (Kan et al. 2004), it compensates, at least partly, for the lack of (P)Cr, which hampers the full assessment of the consequences of Cr deficiency. The recent generation and characterization of an AGAT−/− mouse model provides new opportunities to study the consequences of Cr deficiency (Choe et al. 2012). In contrast to GAMT−/− mice, AGAT−/− mice do not accumulate any guanidino acetate (Choe et al. 2012). As no alternative HEP compounds, such as PGAA, are found in AGAT−/− mice, this mouse provides an ideal model in which to study pure Cr deficiency. Creatine depletion in AGAT−/− mice resulted in significantly reduced total body weight, reduced adiposity and improved glucose tolerance (Choe et al. 2012). The activation of AMP-activated protein kinase by intracellular energy depletion could explain the systemic metabolic phenotype (Choe et al. 2012). However, detailed tissue-specific effects of Cr depletion in skeletal muscle (the tissue containing about 90% of the total body Cr content) have not yet been investigated. The aim of this study was to characterize energy-related metabolic, structural and functional abnormalities caused by systemic Cr deficiency in skeletal muscle of AGAT−/− mice. In addition, we explored to what extent the effects or adaptations could be reversed by Cr supplementation, and used such replenishment to compare the kinetics of Cr accumulation in skeletal muscle with that in brain. Generation and care of the animals as well as all experimental procedures were in accordance with institutional guidelines and national laws for the protection of experimental animals, complied with the regulations of the National Institutes of Health, and were approved by the respective local animal ethics committees (Hamburg, 110/10; Nijmegen, 2007-173). Anesthaesia has been described for the various experiments below. l-Arginine:glycine amidinotransferase knockout mice (AGAT−/−) were generated by a gene-targeting strategy for AGAT deletion (Choe et al. 2012). Heterozygous mice were used for breeding, because homozygous knockout mice were infertile. Chow was essentially Cr free (Sniff, Soest, Germany). Creatine supplementation was achieved by addition of 1% to drinking water or 0.5% Cr to chow. Body weight was determined weekly. To analyse muscle morphology, wild-type (WT) mice and AGAT−/− female mice aged 16–20 weeks, either on a Cr-free diet or on a chow containing Cr (for 12 weeks), were anaesthetized (120 mg kg−1 ketamine and 16 mg kg−1 xylazine) were perfused transcardially with 2% glutaraldehyde in PBS. Tissues were postfixed in 1% OsO4, dehydrated and embedded in Epon. Light microscopic images were recorded after Toluidine Blue staining of semi-thin longitudinal sections from gastrocnemius muscle. Additionally, ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Zeiss EM902 electron microscope (EM). For morphometric analysis of skeletal muscle, fresh muscle tissue from extensor digitorum longus and gastrocnemius muscle (three mice per group) was prepared under anaesthesia (isoflurane 2.5–3% v/v in oxygen) and rapidly frozen after removal of the specimen for histological staining following standard laboratory procedures. Briefly, the specimen was put into mounting medium on a cork plate, with care being taken to yield transverse sections, and put into isopentane (cooled to −160°C). Frozen sections of 8 μm thickness were processed in a standard laboratory cryostat. Per specimen, several sections were mounted on coated slides for histological and histochemical staining procedures. For morphometry, slides routinely stained with haematoxylin and eosin were used. Myocyte diameters were determined in multiple transverse sections of muscle using the AxioVision program (Carl Zeiss Imaging Solutions GmbH, Göttingen, Germany) and calculated by averaging data from five images (Kellner et al. 2009). Maximal grip force of male AGAT−/− knockout mice (−/−), heterozygous (+/−) and wild-type (+/+) mice aged between 15 and 20 weeks was measured using a grip strength meter (TSE-Systems, Bad Homburg, Germany) and compared with mice supplemented with Cr for 12 weeks. Within each group, the mean grip force of each mouse was calculated from 15 appropriate trials. Metabolite levels in hindlimb skeletal muscle were measured non-invasively by 31P and 1H magnetic resonance spectroscopy (MRS). For all magnetic resonance (MR) experiments, mice were anaesthetized with isoflurane (Abott, Cham, Switzerland; induction dose, 4 min at 4–3%; steady state, 1.0–1.8% isoflurane) in a gas mixture of 33/66% O2/air. The steady-state isoflurane dose was adjusted based on the breathing rate, which was monitored continuously using a pneumatic cushion respiratory monitoring system (Small Animal Instruments Inc., Stony Brook, NY, USA) and kept between 80 and 120 breaths min−1. Body temperature was controlled at 37 ± 1°C. The MR experiments were performed in a 200 mm horizontal-bore magnet (Magnex Scientific, Abingdon, UK) interfaced to an MR spectrometer (MR Solutions, Guildford, UK) operating at 300.22 MHz for 1H and at 121.53 MHz for 31P. The magnet was equipped with a gradient insert with gradient strength of 150 mT m−1, rise time of 150 μs and free bore size of 120 mm. The 1H MR measurements of skeletal muscle of the lower hindlimb were performed using a solenoid radio frequency coil positioned at the magic angle with respect to the B0 field (in 't Zandt et al. 1999). Multislice gradient echo images (repetition time (TR) = 250 ms, echo time (TE) = 5 ms, field of view (FOV) 20 mm × 20 mm, 256 × 256 matrix) were used to guide the positioning of 16 μl voxels in the tibialis anterior/extensor digitorus longus region. Localized 1H MR spectra were recorded using stimulated echo acquisition mode (STEAM) (Bottomley, 1987), with variable pulse power and optimized relaxation delays (VAPOR) water suppression (Pfeuffer et al. 1999; Tkáčet al. 1999), TE = 15 ms, TM = 10 ms, TR = 5 s and 128 averages. All 1H spectra were analysed with AMARES (http://sermn02.uab.es/mrui/) using Gaussian line shapes. Absolute levels of total Cr (tCr) and taurine (Tau) in muscle were determined from the water signal of additional 1H spectra acquired without water suppression (eigh averages), assuming a water content of 77% (Sjøgaard & Saltin, 1982) and corrected for T2 relaxation using values determined in a previous study (Renema et al. 2003). The 31P MR spectra in muscle were obtained in AGAT−/− (n = 8) and WT mice (n = 6) by non-localized pulse-acquire experiments (40 μs pulse, α ∼90 deg, TR = 7 s, 256 averages) using a three-turn solenoid radio frequency coil with a diameter of 8 mm (in 't Zandt et al. 2003). Signal integrals were determined by fitting resonances of PCr, inorganic phosphate (Pi), phosphomonoesters and ATP. The phosphomonoesters and Pi line widths were fixed to 0.7 times the line width of β-ATP (experimentally observed in the spectra with the highest signal-to-noise ratio). Metabolite levels were corrected for T1 relaxation and expressed as a ratio with respect to the signal intensity for β-ATP (Kan et al. 2004). We assessed possible differences in absolute ATP tissue concentrations in an additional group of mice (four WT and five AGAT−/− mice) by acquiring one-dimensional Image selected in vivo spectroscopy (ISIS) localized 31P MR spectra (5 mm thick cross-sectional slice of the hindlimb). The localisation of the corresponding muscle volume was obtained from multiple spin echo images (TR/TE = 1500/12 ms, 10 slices, 1 mm slice thickness, FOV = 12 mm × 12 mm, matrix = 128 × 128, two averages) by manual delineation of the cross-sectional muscle area in the ISIS-localized slices. In this way, we could compare the ATP signal intensity per unit muscle volume between WT and AGAT−/− mice. Tissue pH was determined in both tissues from the chemical shift difference (S) between Pi and α-ATP; the frequency of the latter does not shift in the physiological pH range (Moon & Richards, 1973). In brain, 1H MR specra were also recorded using an elliptical 1H surface coil (15 × 11 mm) from a 2.2 mm × 2 mm × 2 mm voxel located partly in thalamus and hippocampus (STEAM, TE = 15 ms, mixing time (TM) = 10 ms, TR = 5 s, 256 averages; Kan et al. 2007). Multislice gradient echo images were acquired in transverse (TR = 4000 ms, TE = 4 ms, 10 slices of 1 mm) and horizontal directions (TR = 4000 ms, TE = 10 ms, 4 slices of 1 mm), avoiding inclusion of ventricles in the volume of interest. The 1H MR brain spectra were analysed with LCModel software to obtain tCr concentrations (Provencher, 1993http://2-provencer.com/pages/lcmodl.shtml). Absolute quantification was performed using the water signal from the unsuppressed spectra (Kan et al. 2007), assuming a mean tissue water content of 78% (in 't Zandt et al. 2004). Relative muscle volumes were determined by drawing the regions of interest (ROIs) around the hindleg muscle in five subsequent cross-sectional gradient echo images (TR = 250 ms, TE = 5 ms, FOV 20 mm × 20 mm, 256 × 256 matrix) positioned 3 mm distal from the knee joint of the hindleg. The surface areas of the five ROIs were multiplied by the 1 mm slice thickness to yield muscle volume over a 5 mm length of the hindleg. Muscle growth upon Cr supplementation was expressed relative to the volume on day 0. To assess the role and capacity of the the PCr–CK system in skeletal muscle, we monitored by 31P MRS the dynamic changes in ATP, PCr, Pi and pH upon ischaemic occlusion of the hindlimb in AGAT−/− and WT mice. Reversible obstruction of blood flow through hindlimb skeletal muscle was accomplished in the MR magnet by clamping the hindlimb above the knee with a diaphragm plate (in 't Zandt et al. 1999). The 31P spectra were acquired with a time resolution of 1.46 min (TR = 1400 ms, 76 averages) for 7 min prior to the ischaemic period, and during 25 min of ischaemia and 16 min of recovery (Kan et al. 2004). The ischaemia experiments in AGAT−/− were performed after a long period of Cr deprivation (i.e. day 0) and at 2 and 21 days of Cr supplementation (n = 5 per group). All metabolite signals acquired before, during or after the ischaemic occlusion were normalized to the mean signal intensity of β-ATP before ischaemia was applied (β-ATP0). For absolute quantification of metabolites, hindlimb muscles (hamstrings) were snap-frozen in liquid nitrogen and stored at −80°C. After freeze drying, 10–15 mg dry muscle tissue was extracted with 1 m perchloric acid. After centrifugation, the supernatant was neutralized with 2 m KHCO3. These extracts were used for spectrophotometric determination of Cr, PCr, ATP, ADP, AMP and lactate (Harris et al. 1974). The biochemically determined metabolite concentrations for each muscle sample were expressed as micromoles per gram dry weight. Hamstring muscles from adult AGAT−/− and WT littermates were removed under isoflurane anaesthesia (5% for >4 min, in a gas mixture of 25/75% O2/air) and the animals were decapitated by cervical dislocation instantly afterwards. The muscle tissue was immediately stored in liquid nitrogen. Mitochondria were isolated from 70–120 mg samples of wet muscle tissue for spectrophotometric determination of mitochondrial enzyme activities of NADH dehydrogenase (complex I, EC 1.6.5.3), succinate dehydrogenase (complex II, EC 1.3.5.1), ubiquinol-cytochrome c reductase (complex III, EC 1.10.2.2), cytochrome c oxidase (complex IV, EC 1.9.3.1), succinate dehydrogenase (SCC, EC 1.3.5.1) and F1F0-ATPase (complex V, EC 3.6.3.14). All the values were expressed as milliunits per unit citrate synthase activity (CS, EC 2.3.3.1). Details of the methods have been described previously (Cooperstein & Lazarow, 1951; Mourmans et al. 1997; Janssen et al. 2007; Jonckheere et al. 2008). Citrate synthase activity was used as a marker for mitochondrial content. The ratio of complex V activity to total ATPase activity was determined in the absence and presence of oligomycin (8 mg ml−1), the specific inhibitor of the F0-part of complex V (Jonckheere et al. 2008). Data are given as means ± SD unless stated otherwise. The following statistical tests were applied: unpaired Student's t test for comparisons between WT and AGAT−/− mice, one-way ANOVA for comparison of WT, AGAT−/− and Cr-supplemented AGAT−/− mice, and two-way ANOVA for multiparametric measurements with Bonferroni's post hoc test using GraphPad Prism (version 4; GraphPad Inc., La Jolla, CA, USA). Light and electron microscopy of hindlimb muscle demonstrated two important morphological abnormalities in muscle fibres of AGAT−/− mice (Fig. 2): first, a large increase in lipid droplets (Fig. 2D, E and G), mainly around the mitochondria (Fig. 2G); and second, electron-dense layers with the typical appearance of crystal structures, located within numerous mitochondria (Fig. 2H). After 12 weeks of Cr supplementation (Fig. 2F), lipid droplets decreased to numbers comparable to those seen in WT mice (Fig. 2A–C), and the intramitochondrial crystal structures were no longer observed. Histopathology also showed a significantly smaller average myocyte diameter in AGAT−/− mice compared with that of WT control animals (Fig. 3A), whereas the AGAT−/− mice that were supplemented with Cr (AGAT−/−+ Cr) had normal-sized myocytes. Hence, the morphological abnormalities seen in the AGAT−/− mice on a Cr-free diet were completely abolished by oral Cr treatment. Photomicrographs of skeletal muscle sections A and D, light microscopic images show Toluidine Blue-stained semi-thin longitudinal sections from hindlimb muscle of wild-type (WT; A) and AGAT−/− mice (D) on a normal Cr-free diet. D, AGAT−/− muscle shows an increased number of lipid droplets (small white dots) when compared with the muscles of the WT control animals. E, G and H, electron microscopic images of hindlimb skeletal muscle of AGAT−/− muscle demonstrate that the lipid droplets were mainly present in close proximity to the mitochondria (G). H, in AGAT−/− muscle multiple mitochondria contain electron-dense bodies between the mitochondrial cristae membranes. B, electron microscopic images of WT mice on a creatine-free diet show normal skeletal muscle for comparison. F, after 12 weeks of Cr supplementation, the number of lipid droplets in the electron microscopic images of the AGAT−/− mice decreases to normal amounts, and the abnormalities in the mitochondria are no longer observed. Creatine supplementation did not reveal any changes in the WT mice. Magnifications: A and D, ×440; and B, C and E–H, ×12,000. Scale bars: (A, D) = 20μm, (B, C, E–H) = 1μm. Reduced muscle volume, myocyte diameter and grip strength in AGAT−/− mice A, myocyte diameters determined from neuropathological analysis. B, muscle force determined by grip strength tests in WT (+/+), and AGAT-deficient knockout (−/−) mice on a Cr-free diet and after 12 weeks of Cr supplementation. C, cross-sectional gradient echo images of hindlimb (TR/TE = 250/5 ms, FOV = 20 mm × 20 mm, 256 × 256 matrix). D, increasing muscle volume of AGAT−/− mice upon Cr supplementation determined from cross-sectional MR images. Values are means ± SEM, n = 5–10 per group. Significant differences compared with all other groups: ***P < 0.001 (Student's unpaired t test). Grip strength of AGAT−/− mice was significantly reduced, by >70%, compared with WT mice (Fig. 3B), while heterozygous littermates did not differ from WT. Creatine supplementation improved grip strength in all groups; however, the gain was statistically significant only in the AGAT−/− mice, which showed a complete recovery of strength to WT levels. The AGAT−/− mice were hypotonic (hanging down when lifted by their tail), but muscle tone and habitus normalized upon Cr supplementation. Body weight was significantly reduced in Cr-depleted AGAT−/− mice (19.7 ± 2.2 g, n = 8) compared with WT animals (37.8 ± 7.1 g, n = 5) and was accompanied by severe muscle atrophy. Cross-sectional gradient-echo MR images of hindlimb muscles demonstrate a reduction of relative muscle volume to 34 ± 9% (n = 5) compared with WT (100 ± 21%, n = 4; Fig. 3C). The decreased muscle volumes are consistent with the decreased myocyte diameters described above (see ‘Morphology’). The atrophy was apparently not confined to the leg muscles; thoracolumbar kyphosis in AGAT−/− mice suggests additional dysfunction of the postural and paraspinal muscles. Creatine supplementation for 3 months increased the body weight of AGAT−/− mice up to 56%, while age-matched WT mice showed an increase of only 19.3 ± 4.7% over that period, which is not significantly different from WT animals on a normal diet (15.3 ± 2.2%). In contrast, age-matched AGAT−/− mice showed no increase in body weight during this period. The body weight of heterozygous mice did not differ from that of WT animals regardless of diet. Besides the normalization of body weight and composition (i.e. water, fat and lean mass) during Cr supplementation (Choe et al. 2012), skeletal muscle volume also recovered, as demonstrated by the increase in cross-sectional muscle areas and amelioration of kyphosis (Fig. 3D). In vivo 1H MR spectra of skeletal muscle of AGAT−/− mice demonstrated virtually complete absence of the Cr signals at 3.0 and 3.9 p.p.m., while taurine levels were not significantly different from those of WT mice (Fig. 4A). As expected, AGAT−/− muscles did not show PCr signals at 0 p.p.m. in 31P MR spectra either (Fig. 4B). Importantly, these spectra did not reveal any other phosphorylated guanidine compound that could replace PCr in its ATP-buffering role, such as phosphorylated forms of guanidinoacetate or arginine. Upon Cr supplementation, normal PCr and tCr signals were observed (see Table 1). Intracellular pH, as calculated from the chemical shift difference between α-ATP and Pi, was not statistically different between the two groups. Interestingly, the lack of Cr in skeletal muscle of the mutants was accompanied by significant elevation of the Pi/β-ATP ratio, approximately fourfold compared with WT levels (Table 1), as a consequence of either low ATP concentration or high Pi concentration, or both. To resolve this, we determined the ratio of β-ATP signal intensity to muscle volume over five slices, which was significantly reduced in the AGAT−/− mice (63 ± 8% of WT levels). Biochemical analysis confirmed decreased ATP concentrations by 53% in the AGAT−/− muscle (Table 1). The decreased ATP levels imply that absolute Pi concentrations are increased by approximately twofold in AGAT−/− muscle compared with WT. Biochemical determinations of tissue metabolite concentrations confirmed the nearly complete absence of PCr in AGAT−/− hamstring muscle, demonstrating a ∼87% depletion in Cr content compared with WT. Very similar PCr/ATP ratios in WT muscle were calculated from biochemical determinations (3.0 ± 0.4) and in vivo MR spectra (3.2 ± 0.1). Biochemically determined concentrations of ADP, AMP and lactate in muscle were not significantly different between WT and AGAT−/− mice, whereas ATP/ADP and ATP/AMP ratios were both much lower in the AGAT−/− muscle (Table 1). Magnetic resonance spectra of muscle obtained from WT mice (top) and AGAT−/− mice on a Cr-free diet A 1H MR spectrum (A) obtained from a 16 μl voxel and a non-localized 31P MR spectrum (B) obtained in hindlimb tissue of WT (upper spectra) and AGAT−/− mice (lower spectra) on a Cr-free diet. The unlocalized 31P AGAT−/− spectrum was multiplied by four to increase visibility, which was a direct consequence of the severe reduction in muscle volume. Note the absence of total creatine (tCr) and phosphocreatine (PCr) and the relatively large inorganic phosphate (Pi) signal in the AGAT−/− muscle. The increased Pi and decreased ATP concentrations suggested adaptive changes in oxidative phosphorylation in AGAT−/− muscle. We therefore measured respiratory chain activities in isolated mitochondria of muscle tissue. Enzyme activities of complex II and complex V, total ATPase and citrate synthase were significantly elevated per gram of wet muscle tissue (Table 2). Given that CS is commonly used as a marker for mitochondrial mass, the elevation of its activity in AGAT−/− muscle (170% of WT levels) indicates an increased mitochondrial content. The ratio of F-type ATPase (complex V) to total ATPase activity was comparable between the two groups, hence not only is F-type ATPase increased by ∼90%, but total ATPase activity per mitochondrial content (mg wet weight) is equally increased. Using CS activity as a marker for mitochondrial content, activities of complexes III and IV per mitochondrial content in AGAT−/− muscle were significantly decreased by 36 and 27%, respectively (Table 2). A similar decrease was seen in complex I (38%), although it did not reach statistical significance. This implies that the H+-pumping respiratory enzymes are downregulated in mitochondria of AGAT−/− muscle, while other respiratory enzyme activities were not significantly different from WT muscle. Creatine-supplemented AGAT−/− mice demonstrated a rapid accumulation of Cr in skeletal muscle (Fig. 5A). The tCr signal intensities of 1H MR spectra from tibialis anterior/extensor digitorum longus muscle reached normal WT levels within 1 day of supplementation, whereas the accumulation in brain was much slower (Fig. 5B). In the hypothalamic/hippocampal region of AGAT−/− mice, tCr levels gradually increased to WT levels over about 20 days. Given that we previously studied Cr supplementation in GAMT−/− mice in the same experimental set-up and with the same Cr dosing protocol (Kan et al. 2007), we can directly compare treatment responses between these two models of Cr deficiency. Two differences are apparent in Cr accumulation (Fig. 5A and B). Creatine accumulation in AGAT−/− muscle occurs faster than in GAMT−/− animals, but levels off after 2 days. In contrast, skeletal muscle tCr levels in GAMT−/− mice increased up to about 4 weeks, eventually reaching higher levels than in AGAT−/− mice. However, there is no apparent difference in the kinetics of cerebral Cr accumulation between the two models. Metabolic changes in AGAT−/− and GAMT−/− mice during Cr supplementation A and B, changes in total creatine (tCr) concentrations were obtained from 1H spectra of muscle from a 16 μl voxel (STEAM) in the lower limb tibialis anterior/extensor digitorum longus (A) and an 8.8 μl voxel in the hypothalamic/hippocampal region of the brain (B). Total Cr levels in AGAT−/− mouse muscle (filled circles) were compared with tCr levels obtained in triceps surae in GAMT−/− mice (grey circles; the GAMT−/− data were used with permission from Kan et al. 2007). C–E, changes in PCr/β-ATP (C) and Pi/β-ATP signal ratios (D) and taurine concentration (E) in hindleg muscle of AGAT−/− mice during Cr administration. The ratios were determined from unlocalized 31P MR spectra. Data are means ± SEM, n ≥ 3 per time point. Most Cr taken up by muscle is immediately phosphorylated, as shown by the near-instantaneous increase of PCr/β-ATP ratios upon Cr supplementation (Fig. 5C). An initi" @default.
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• W1598394864 date "2013-01-01" @default.
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• W1598394864 title "Disturbed energy metabolism and muscular dystrophy caused by pure creatine deficiency are reversible by creatine intake" @default.
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