Dose-dependent effects of leucine supplementation on preservation of muscle mass in cancer cachectic mice.

Cancer cachexia, which is characterized by muscle wasting, is associated with increased morbidity and mortality. Because muscle protein synthesis may be increased and protein breakdown reduced by leucine supplementation, we used the C26 tumor-bearing cachectic mouse model to assess the effects of dietary supplementation with leucine on muscle weight and the markers of muscle protein breakdown (mRNA of atrogin and murf). Male CD2F1 mice were subcutaneously inoculated with tumor cells (tumor-bearing mice; TB) or were sham injected (control; C). They were fed standard diets or diets supplemented with leucine [1 gr (TB1Leu) or 8 gr (TB8Leu) supplemented leucine per kg feed]; TB and C received 8.7% Leu/g protein, TB1Leu received 9.6% Leu/g protein and TB8Leu received 14.6 Leu/g protein. After 21 days, the following were determined: body weights, plasma amino-acid concentrations, tumor size and muscle mass of the gastrocnemius (mG), tibialis anterior (mTA), extensor digitorum longus (mEDL) and soleus (mS) muscles. In tumor-bearing (TB) mice, carcass and skeletal muscle masses decreased, and levels of atrogin and murf mRNA in the mEDL increased. Muscle-mass loss was counteracted dose-dependently by leucine supplementation: relative to TB, the mass of the mG was +23% in TB8Leu, and +22% in mTA (p<0.05). However, leucine supplementation did not change atrogin and murf mRNA levels. Total plasma amino acid concentrations increased in TB, especially for taurine, lysine, arginine and alanine (p<0.05). Leucine supplementation attenuated the increase in total plasma amino-acid concentrations (p<0.05). Irrespective of changes in muscle protein breakdown markers, leucine supplementation reduced muscle wasting in tumor-bearing cachectic mice and attenuated changes in plasma amino acids.

Dietary supplementation with a specific combination of high protein, leucine, and fish oil improves muscle function and daily activity in tumour-bearing cachectic mice.

Cancer cachexia is characterised by metabolic alterations leading to loss of adipose tissue and lean body mass and directly compromises physical performance and the quality of life of cancer patients. In a murine cancer cachectic model, the effects of dietary supplementation with a specific combination of high protein, leucine and fish oil on weight loss, muscle function and physical activity were investigated. Male CD2F1 mice, 6-7 weeks old, were divided into body weight-matched groups: (1) control, (2) tumour-bearing, and (3) tumour-bearing receiving experimental diets. Tumours were induced by s.c. inoculation with murine colon adenocarcinoma (C26) cells. Food intake, body mass, tumour size and 24 h-activity were monitored. Then, 20 days after tumour/vehicle inoculation, the animals were killed and muscle function was tested ex vivo. Tumour-bearing mice showed reduced carcass, muscle and fat mass compared with controls. EDL muscle performance and total daily activity were impaired in the tumour-bearing mice. Addition of single nutrients resulted in no or modest effects. However, supplementation of the diet with the all-in combination of high protein, leucine and fish oil significantly reduced loss of carcass, muscle and fat mass (loss in mass 45, 52 and 65% of TB-con, respectively (P<0.02)) and improved muscle performance (loss of max force reduced to 55-64% of TB-con (P<0.05)). Moreover, total daily activity normalised after intervention with the specific nutritional combination (50% of the reduction in activity of TB-con (P<0.05)). In conclusion, a nutritional combination of high protein, leucine and fish oil reduced cachectic symptoms and improved functional performance in cancer cachectic mice. Comparison of the nutritional combination with its individual modules revealed additive effects of the single components provided.

Direct effects of doxorubicin on skeletal muscle contribute to fatigue.

Chemotherapy-induced fatigue is a multidimensional symptom. Oxidative stress has been proposed as a working mechanism for anthracycline-induced cardiotoxicity. In this study, doxorubicin (DOX) was tested on skeletal muscle function. Doxorubicin induced impaired ex vivo skeletal muscle relaxation followed in time by contraction impediment, which could be explained by DOX-induced changes in Ca(2+) responses of myotubes in vitro. The Ca(2+) responses in skeletal muscle, however, could not be explained by oxidative stress.

Increased muscle fatigability in GLUT-4-deficient mice.

GLUT-4 plays a predominant role in glucose uptake during muscle contraction. In the present study, we have investigated in mice whether disruption of the GLUT-4 gene affects isometric and shortening contractile performance of the dorsal flexor muscle complex in situ. Moreover, we have explored the hypothesis that lack of GLUT-4 enhances muscle fatigability. Isometric performance normalized to muscle mass during a single tetanic contraction did not differ between wild-type (WT) and GLUT-4-deficient [GLUT-4(-/-)] mice. Shortening contractions, however, revealed a significant 1.4-fold decrease in peak power per unit mass, most likely caused by the fiber-type transition from fast-glycolytic fibers (IIB) to fast-oxidative fibers (IIA) in GLUT-4(-/-) dorsal flexors. In addition, the resting glycogen content was significantly lower (34%) in the dorsal flexor complex of GLUT-4(-/-) mice than in WT mice. Moreover, the muscle complex of GLUT-4(-/-) mice showed enhanced susceptibility to fatigue, which may be related to the decline in the muscle carbohydrate store. The significant decrease in relative work output during the steady-state phase of the fatigue protocol suggests that energy supply via alternative routes is not capable to compensate fully for the lack of GLUT-4.

Murine muscles deficient in creatine kinase tolerate repeated series of high-intensity contractions.

Murine muscles lacking both mitochondrial (Mi-CK) and cytoplasmic (MM-CK) creatine kinase (CK-/-) show depressed mechanical performance in association with low muscle ATP and enhanced IMP content. The aims of the present study were to elucidate the possible role of low ATP and high IMP content in impairment of mechanical performance in CK-/- mice and to establish whether CK-/- muscles are able to sustain repeated series of high-intensity contractions. The dorsal flexors of CK-/- and control mice were subjected in situ to two series of 12 tetanic contractions using a custom-made mouse isometric dynamometer. The muscle content of high-energy phosphates was analysed by HPLC. ATP content declined from 20.6+/-1.9 to 15.5+/-2.4 micromol x g(-1) dry weight (d.w.); IMP content increased from 1.2+/-0.4 to 2.4+/-1.1 micromol x g(-1) d.w. during the first contraction series in CK-/- muscle. Despite these unfavourable changes, maximal torque developed during the first contraction of either series did not differ, indicating that the altered content of ATP and IMP does not play a decisive role in impaired mechanical performance in CK-/- mice. The relative decline in torque during the two series did not differ in CK-/- (-20.4+/-6.6 vs. -23.8+/-9.9%). In contrast, wild-type (WT) muscles showed a significantly more pronounced decline during the second series (-12.3+/-7.4 vs. -20.1+/-6.8%). Muscle ATP and IMP content did not change in CK-/-, whereas in WT IMP content increased significantly during the second contraction series. These findings indicate that CK-/- tolerate repeated series of high-intensity contractions better than WT, while in CK-/- muscle an additional source of energy is mobilised to regenerate ATP during the second series.

Impaired muscular contractile performance and adenine nucleotide handling in creatine kinase-deficient mice.

Creatine kinase (CK) forms a small family of isoenzymes playing an important role in maintaining the concentration of ATP and ADP in muscle cells. To delineate the impact of a lack of CK activity, we studied contractile performance during a single maximal tetanic contraction and during 12 repeated tetanic contractions of intact dorsal flexors of CK knockout (CK(-/-)) mice. To investigate the effect on ATP regeneration, muscular high-energy phosphate content was determined at rest, immediately after the contraction series, and after a 60-s recovery period. Maximal torque of the dorsal flexors was significantly lower in CK(-/-) mice than in wild-type animals, i.e., 23.7 +/- 5.1 and 33.3 +/- 6.8 mN. m. g(-1) wet wt, respectively. Lower muscle ATP (20.1 +/- 1.4 in CK(-/-) vs. 28.0 +/- 2.1 micromol/g dry wt in controls) and higher IMP (1.2 +/- 0.5 in CK(-/-) vs. 0.3 +/- 0.1 micromol/g dry wt in controls) levels at the onset of contraction may contribute to the declined contractility in CK(-/-) mice. In contrast to wild-type muscles, ATP levels could not be maintained during the series of 12 tetanic contractions of dorsal flexors of CK(-/-) mice and dropped to 15.5 +/- 2.4 micromol/g dry wt. The significant increase in tissue IMP (2.4 +/- 1.1 micromol/g dry wt) content after the contraction series indicates that ATP regeneration through adenylate kinase was not capable of fully compensating for the lack of CK. ATP regeneration via the adenylate kinase pathway is a likely cause of reduced basal adenine nucleotide levels in CK(-/-) mice.

In situ assessment of shortening and lengthening contractile properties of hind limb ankle flexors in intact mice.

The availability of animal models with disrupted genes has increased the need for small-scale measurement devices. Recently, we developed an experimental device to assess in situ mechanical properties of isometric contractions of intact muscle complexes of the mouse. Although this apparatus provides valuable information on muscle mechanical performance, it is not appropriate for determining contractile properties during shortening and lengthening contractions. In the present study we therefore developed and evaluated an experimental apparatus for assessment of shortening and lengthening contractile properties of intact plantar and dorsal flexors of the mouse. The current through a custom-built, low-inertia servomotor was measured to assess contractile muscular torque ranging from -50 to mN.m. Evaluation of the fixation procedure of the animal to the apparatus via 3-D monitoring of the muscle-tendon complex length showed that the additional shortening in length due to a contraction with maximal torque output has only minor effects on the measured torque. Furthermore, misalignment of the axis of rotation of the apparatus relative to the axis of rotation in the ankle joint, i.e. eccentricity, during a routine experiment was estimated to be less than 1.0 mm and hence did not influence the measured torque output under our experimental conditions. Peak power per unit muscle mass (mean +/- SD) of intact dorsal and plantar flexors was 0.27 +/- 0.02 and 0.19 +/- 0.03 W.g-1, respectively. The angular velocity at maximal peak power generated by the dorsal flexor complex and the plantar flexor complex was 1100 +/- 190 and 700 +/- 90 degrees.s-1, respectively.

Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration.

Hypoxia stimulates angiogenesis through the binding of hypoxia-inducible factors to the hypoxia-response element in the vascular endothelial growth factor (Vegf) promotor. Here, we report that deletion of the hypoxia-response element in the Vegf promotor reduced hypoxic Vegf expression in the spinal cord and caused adult-onset progressive motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis. The neurodegeneration seemed to be due to reduced neural vascular perfusion. In addition, Vegf165 promoted survival of motor neurons during hypoxia through binding to Vegf receptor 2 and neuropilin 1. Acute ischemia is known to cause nonselective neuronal death. Our results indicate that chronic vascular insufficiency and, possibly, insufficient Vegf-dependent neuroprotection lead to the select degeneration of motor neurons.

Accurate assessment of in situ isometric contractile properties of hindlimb plantar and dorsal flexor muscle complex of intact mice.

An isometric torque sensor for measuring in situ contractions of plantar or dorsal flexors of intact mouse hindlimb has been developed and evaluated. With this device, muscle torque can be accurately measured within the range of -14 mN.m to +14 mN.m. Special attention was paid to fixation of the mouse hindlimb to the measurement device. Halothane-anaesthetized Swiss wild-type mice were positioned on the thermostatic measurement platform, and fixated with a hip and foot fixation system. The novel fixation unit was evaluated by measuring knee and ankle displacements during a contraction. A mathematical muscle model was used to quantify the effects of these displacements on the contractile parameters. Measured ankle and knee displacement, due to non-absolute fixation. resulted in a calculated muscle fibre shortening of 2.5%. Simulations of a contraction with this degree of fibre shortening, using the mathematical muscle model, showed only minor effects on maximal torque generation and the temporal parameters (half-relaxation time and 10-50% rise time). Furthermore, we showed that muscle torque in our set-up is hardly affected by eccentricity between ankle and measurement axis. Measured tetanic muscle torques of intact dorsal and plantar flexors were 3.2+/-0.4 mN.m and 11.8+/-1.6 mN.m, respectively. The half-relaxation time of plantar flexors was significantly higher than that of dorsal flexors (12.9+/-2.7 ms versus 8.8+/-1.2 ms), whereas the 10-50% rise time was longer in plantar (14.9+/-0.6 ms) than in dorsal (11.8+/-2.0 ms) flexors.