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A hallmark of glycogen storage disease type II, caused by defective alpha-glucosidase (AGLU) activity, is a progressive decline in muscle performance. The objective of this study was to determine the relative contribution to this decline in muscle performance of (1) decline in muscle mass; (2) decline in muscle protein content per unit mass; and (3) accumulation of glycogen. To this end, isometric torque and power in AGLU(-/-) mice at 7, 13, and 20 months were assessed in situ. Power (approximately 24 mW) and torque (approximately 2.45 Nmm) did not change with age in control animals, but declined significantly in AGLU(-/-) mice, in the three age groups. No decline in protein content per unit muscle mass was observed. Muscle atrophy explained one third of the decline in muscle performance; the remaining part was attributed to a decrease in muscle quality--a decrease in mechanical performance per unit muscle mass. Mechanical effects of glycogen inclusions could not fully explain this decrease. Additional factors must therefore play a role.

A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections.Schaart G, Hesselink RP, Keizer HA, van Kranenburg G, Drost MR, Hesselink MK.Department of Movement Sciences, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands.Simultaneous analyses of glycogen in sections with other subcellular constituents within the same section will provide detailed information on glycogen deposition and the processes involved. To date, staining protocols for quantitative glycogen analyses together with immunofluorescence in the same section are lacking. We aimed to: (1) optimise PAS staining for combination with immunofluorescence, (2) perform quantitative glycogen analyses in tissue sections, (3) evaluate the effect of section thickness on PAS-derived data and (4) examine if semiquantitative glycogen data were convertible to genuine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of glycogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r2=0.993, P

Effect of testosterone and endurance training on glycogen metabolism in skeletal muscle of chronic hyperglycaemic female rats.van Breda E, Keizer H, Kuipers H, Kranenburg G.Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht University, Maastricht, The Netherlands. vanbreda@bw.unimaas.nlOBJECTIVES: To investigate in glycolytic and oxidative muscles of trained (nine weeks) and untrained hyperglycaemic female rats the effect of hyperandrogenicity and/or endurance training on energy metabolic properties. METHODS: Glycogen content and activity of muscle enzymes with regulatory functions in glycogen synthesis were examined. RESULTS: Testosterone treatment increased glycogen content of extensor digitorum longus (EDL) and soleus muscles of hyperglycaemic sedentary (18% and 84% respectively) and hyperglycaemic trained (7% and 16% respectively) rats. In both types of muscle of the hyperglycaemic testosterone treated exercised subgroup, less depletion of glycogen was found than in the untreated group (38% and 87% for EDL and soleus respectively). CONCLUSIONS: The mechanisms by which training and/or hyperandrogenism alone or in combination elicits their specific effects are complex. Differences in sex, surgery, levels of hormones administered, and exercise model used may be the main reasons for the observed discrepancies. Conclusions from the results: (a) hyperandrogenism is not a primary cause of the development of insulin resistance; (b) glycogen content of slow and fast twitch muscle is increased by training through increased glycogen synthase activity. The most plausible explanation for differences between different muscle fibre types is the different levels of expression of androgen receptors in these fibres. Hyperandrogenicity therefore acts on energy metabolic variables of hyperglycaemic animals by different mechanisms in glycolytic and oxidative muscle fibres

It has been hypothesized that exercise-related hypo-estrogenemia occurs as a consequence of increased competition of catecholestrogens (CE) for catechol-O-methyltransferase (COMT). This may result in higher norepinephrine (NE) concentrations, which could interfere with normal gonadotropin pulsatility. The present study investigates the effects of training on CE responses to acute exercise stress. Nine untrained eumenorrheic women (mean percentage of body fat +/-SD: 24.8 +/- 3.1%) volunteered for an intensive 5-day training program. Resting, submaximal, and maximal (tmax) exercise plasma CE, estrogen, and catecholamine responses were determined pre- and post training in both the follicular (FPh) and luteal phase (LPh). Acute exercise stress increased total primary estrogens (E) but had little effect on total 2-hydroxyestrogens (2-OHE) and 2-hydroxyestrogen-monomethylethers (2-MeOE) (= O-methylated CE after competition for catechol-O-methyltransferase). This pattern was not significantly changed by training. However, posttraining LPh mean (+/-SE) plasma E, 2-OHE, and 2-MeOE concentrations were significantly lower (P < 0.05) at each exercise intensity (for 2-OHE: 332 +/- 47 vs. 422 +/- 57 pg/mL at tmax; for 2-MeOE: 317 +/- 26 vs. 354 +/- 34 pg/mL at tmax). Training produced opposite effects on 2-OHE:E ratios (an estimation of CE formation) during acute exercise in the FPh (reduction) and LPh (increase). The 2-MeOE:2-OHE ratio (an estimation of CE activity) showed significantly higher values at tmax in both menstrual phases after training (FPh: +11%; LPh: +23%; P < 0.05). After training, NE values were significantly higher (P < 0.05). The major findings of this study were that: training lowers absolute concentrations of plasma estrogens and CE; the acute exercise challenge altered plasma estrogens but had little effect on CE; estimation of the formation and activity of CE suggests that formation and O-methylation of CE proportionately increases. These findings may be of importance for NE-mediated effects on gonadotropin release.

De Crée, Carl, Peter Ball, Bärbel Seidlitz, Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer. Plasma 2-hydroxycatecholestrogen responses to acute submaximal and maximal exercise in untrained women. J. Appl. Physiol. 82(1): 364–370, 1997.—Exercise-induced menstrual problems are accompanied by an increase in catecholestrogen (CE) formation. It has been hypothesized that hypoestrogenemia may be secondary to an increased turnover from estrogens to CE, which then may disrupt luteinizing hormone release. In addition, the strong affinity of CE for the catecholamine-deactivating enzyme catechol- O-methyltransferase (COMT) has led to speculations about their possible role in safeguarding norepinephrine from premature decomposition during exercise. We investigated whether acute exercise on a cycle ergometer produces any changes in CE homeostasis. Nine untrained eumenorrheic women (body fat, 24.8 ± 3.1%) volunteered for this study. Baseline plasma CE averages for total 2-hydroxyestrogens (2-OHE) were 218 ± 29 (SE) pg/ml during the follicular phase (FPh) and 420 ± 58 pg/ml during the luteal phase (LPh). 2-Methoxyestrogens (2-MeOE) measured 257 ± 17 pg/ml in the FPh and 339 ± 39 pg/ml in the LPh. During incremental exercise, total estrogens (E) increased, but 2-OHE and 2-MeOE levels did not significantly change in either phase. The 2-OHE/E ratio (measure of CE turnover) decreased during exercise in both menstrual phases, whereas the 2-MeOE/2-OHE ratio (correlates with COMT activity) did not significantly change. These findings suggest that there is insufficient evidence to conclude that brief incremental exercise in untrained eumenorrheic females acutely produces increased CE formation.

Physical exercise training is a powerful tool to maintain or improve mitochondrial density and function (mitochondrial capacity). This study aims to determine whether mitochondrial capacity is also associated with habitual physical activity in daily life (PADL). The capacity of classic markers for mitochondrial density, i.e., the capacity of citrate synthase (CS) and succinate dehydrogenase (SDH), as well the capacity of cytochrome c oxidase (COX) and β-hydroxyacyl-CoA dehydrogenase (HAD), was determined in homogenized muscle biopsy samples obtained from the vastus lateralis muscle of nonexercising healthy young (age 20 ± 2 yr) subjects (31 women, 7 men). PADL was measured during two periods of 14 days using a triaxial accelerometer for movement registration. CS, SDH, and COX were positively associated with PADL [ P < 0.05, R = 0.36, 95% confidence interval (CI): 1.3·10−4 to 2.2·10−3; P < 0.05, R = 0.39, 95% CI: 1.1·10−5 to 9.9·10−5; and P < 0.05, R = 0.33, 95% CI: 7.5·10−6 to 3.6·10−4, respectively], and HAD tended to correlate positively with PADL ( P = 0.06, R = 0.31, 95% CI: −2.2·10−5 to 1.1·10−3). The population was subsequently stratified based on the intensity of the activities performed. CS was only associated with PADL in subjects spending more time on high-intensity physical activity, whereas HAD was only associated with PADL in subjects spending less time on low intensity physical activity. We are the first to report that even within the range of normal daily life activities, mitochondrial capacity is positively associated with the level of habitual physical activity in daily life. Thus an active lifestyle may help to maintain or improve mitochondrial capacity.

An MR-compatible device for the in situ assessment of isometric contractile performance of mouse hind-limb ankle flexors.Drost MR, Heemskerk AM, Strijkers GJ, Dekkers EC, Van Kranenburg G, Nicolay K.Dept. Movement Sciences, Nutrition and Toxicology Research Institute Maastricht, Maastricht University, P.O. Box 616, 6200 MD, Maastricht, The Netherlands, Maarten.Drost@bw.unimaas.nlThe goal of the present study was to develop and evaluate an isometric dynamometer for measuring mouse ankle flexor torque after electric stimulation of the nerve. The dynamometer was to be used within an magnetic resonance (MR) apparatus and should require minimal surgical intervention. To quantify the effect of the magnetic field on contractile parameters, measurements were performed both outside and inside the MR apparatus. The effect of magnetic field gradient switching that accompanies rapid MR scanning was tested also. The set-up required no surgical intervention except for chronic implantation of an electrode. The dynamometer has a high mechanical frequency response (270 Hz). Measured muscle strengths were identical outside and inside the MR scanner. However, during fast magnetic field gradient switching, the variability increased and the measured strength decreased slightly (7%). The noise level of the dynamometer (0.02-0.03 N.mm) was low compared with the strength of the dorsal flexors (2 N.mm). Fast gradient switching increased the noise level (0.07 N.mm). The dynamometer had no observable adverse effects on the quality of the MR images of the mouse hind limb. We conclude that the dynamometer enables accurate measurements of mechanical muscle performance during exercise protocols within an MR apparatus under physiological conditions

De Crée, Carl, Peter Ball, Bärbel Seidlitz, Gerrit Van Kranenburg, Peter Geurten, and Hans A. Keizer.Effects of a training program on resting plasma 2-hydroxycatecholestrogen levels in eumenorrheic women. J. Appl. Physiol. 83(5): 1551–1556, 1997.—Catecholestrogens (CE) represent a major metabolic pathway in estrogen metabolism. Previous information on CE and training is limited to two cross-sectional studies that did not involve standardized training. Our purpose, by means of a prospective design, was to evaluate the effects of a brief, exhaustive training program on resting plasma concentrations of 2-hydroxy CE. The experimental design spanned two menstrual cycles: a control cycle and a training cycle. The subjects were nine previously untrained, eumenorrheic women [body fat: 24.8 ± 1.0 (SE) %]. Data were collected during the follicular (FPh) and the luteal phases (LPh). Posttraining FPh and LPh tests were held the day after the last day of a 5-day period of training on a cycle ergometer. Total 2-hydroxyestrogens (2-OHE) averaged 200 ± 29 pg/ml during the FPh and 420 ± 54 pg/ml during the LPh ( P < 0.05). Levels of total 2-methoxyestrogens (2-MeOE) were 237 ± 32 pg/ml during the FPh and 339 ± 26 pg/ml during the LPh ( P< 0.05). After training, although the plasma levels of 2-OHE significantly decreased (−21%; P < 0.05) during the LPh, the actual CE formation (as estimated from the 2-OHE-to-total estrogens ratio) increased (+29%; P < 0.05). CE activity, as expressed by the 2-MeOE-to-2-OHE ratio, showed significantly higher values in both phases (FPh, +14%; LPh, +13%; P < 0.05). At the same time, resting levels of norepinephrine (NE) were increased by 42% ( P < 0.05). CE strongly inhibit biological decomposition of NE by catechol- O-methyltransferase (COMT). Results of the present study suggest that, in response to training, CE are increasingly competing with the enzyme COMT, thus preventing premature NE deactivation.

The effects of training and/or testosterone treatment on glycogen content and the activities of glycogen synthase, glycogen phosphorylase, and fructose-6-phosphate kinase were studied in extensor digitorum longus (EDL) and soleus muscles of intact adult female rats. One group of rats remained sedentary, whereas another group was trained for 7 weeks. Thereafter, both the sedentary and trained rats were subdivided into two control and four testosterone-treated subgroups. Testosterone was administered by a silastic implant. Training was continued for 2 weeks. On the final day of the experiment rats from one trained control and one trained testosterone-treated subgroup ran for 60 min submaximally. Upon testosterone treatment of sedentary rats the glycogen concentration was not changed. However, in the soleus, but not in the EDL, the glycogen content was increased by training (P