Androgens regulate cell growth, lysosomal hydrolases and mitochondrial cytochrome c oxidase in mouse aorta

The aorta in male mice shows higher activities of several lysosomal hydrolases and of cytochrome c oxidase, an inner mitochondrial membrane enzyme, than in female mice. Orchiectomy abolishes this sex difference, whereas testosterone administration induces an accretion of RNA and protein and elevated activities of lysosomal hydrolases and cytochrome c oxidase. However, the outer mitochondrial membrane enzyme monoamine oxidase is unaffected by sex, orchiectomy or testosterone. Thus, androgens regulate cell growth and enzymes associated with lysosomes and the inner mitochondrial membrane.     

Effect of sciatic nerve crush on enzyme activities of skeletal muscles of the rat.

Weight loss and reduction in specific activity of cytochrome oxidase of both red (soleus) and white muscles (gastrocnemius and plantaris) of the rat was greatest 5 days after sciatic nerve crush (p less than 0.001) and then became minimal. In neither was there a significant, concomitant loss of protein. By 19 days, the specific activity of cytochrome oxidase was the same in both muscle types. The specific activity of lactate dehydrogenase was reduced significantly (p less than 0.001) in the white muscles, to a value approaching that of the red soleus by 19 days postoperatively, but remained unaltered in the soleus. Nerve crush is proposed as a model experimental system for studying neural regulation of skeletal muscle metabolism.     

Changes in lysosomal enzyme activities in exercise-induced cardiac hypertrophy of mice

The activities of several lysosomal enzymes were assayed in control and in exercise-hypertrophied cardiac muscle of mice (Mus musculus). The repeated running program increased the activity of beta-glucuronidase (16.1%) in mouse cardiac muscle. Decreased activities of beta-N-acetylglucosaminidase (10.8%), acid ribonuclease (10.7%), and arylsulphatase (14.2%) were observed in the hypertrophied myocardium. The activities of acid deoxyribonuclease, cathepsin C, cathepsin D, and p-nitrophenylphosphatase as well as the activities of citrate synthase and cytochrome c oxidase, mitochondrial enzymes, were unaffected in cardiac muscle. We suggest that lysosomal enzyme responses are selective and highly different in physiologically and pathologically induced cardiac hypertrophies.     

Lack of myostatin alters intermyofibrillar mitochondria activity, unbalances redox status, and impairs tolerance to chronic repetitive contractions in muscle

Loss of myostatin (mstn) function leads to a decrease in mitochondrial content, a reduced expression of cytochrome c oxidase, and a lower citrate synthase activity in skeletal muscle. These data suggest functional or ultrastructural mitochondrial abnormalities that can impact on muscle endurance characteristics in such phenotype. To address this issue, we investigated subsarcolemmal and intermyofibrillar (IMF) mitochondrial activities, skeletal muscle redox homeostasis, and muscle fiber endurance quality in mstn-deficient mice [mstn knockout (KO)]. We report that lack of mstn induced a decrease in the coupling of IMF mitochondria respiration, with significantly higher basal oxygen consumption. No lysis of mitochondrial cristae or excessive swelling were observed in mstn KO mice compared with wild-type (WT) mice. Concerning redox status, mstn KO gastrocnemius exhibited a significant decrease in lipid peroxidation levels (-56%; P < 0.01 vs. WT) together with a significant upregulation of the antioxidant glutathione system. In contrast, superoxide dismutase and catalase activities were altered in mstn KO, gastrocnemius and soleus with a reduction of up to 80% compared with WT animals. The force production observed after contractile endurance test was significantly lower in extensor digitorum longus and soleus muscles of mstn KO mice compared with the controls (17 ± 3 and 36 ± 5% vs. 28 ± 4 and 56 ± 5%, respectively, P < 0.05). Together, these findings indicate that, besides an increased skeletal muscle mass, genetic mstn inhibition has differential effects on redox homeostasis and mitochondrial function that would have functional consequences on muscle response to endurance exercise.     

Glucocorticoids and aspirin inhibit and cyproterone acetate enhances the androgenic response in mouse kidney

Glucocorticoids and aspirin antagonize the androgenic response in mouse kidney, but not in ventral prostate or seminal vesicles. These agents impeded the testosterone-mediated increase in kidney weight, cytochrome c oxidase, and lysosomal hydrolases and urinary excretion of lysosomal hydrolases and proteins. They also attenuated the testosterone-induced decrease in enzyme latency and membrane stability of kidney lysosomes. In contrast, the antiandrogen cyproterone acetate is weakly androgenic in kidney and potentiates testosterone-induced lysosomal enzymuria and proteinuria (synandrogenic effect).     

Effects of thyrotoxicosis on mitochondrial enzymes of rat soleus.

Cytochrome oxidase, glycerol-3-phosphate dehydrogenase, and succinate dehydrogenase were measured in mitochondrial fractions obtained from rat soleus muscle of control and 8 week T3 + T4 treated animals. Under these conditions of prolonged treatment, there is a five-fold increase in the specific activities of both cytochrome oxidase and glycerole-3-phosphate dehydrogenase. Significant increases in total cellular mitochondrial content and enzyme activities were observed in T3 + T4 treated animals as compared to controls. These results indicate that thyrotoxicosis can induce selective changes in mitochondrial enzymes in slow twitch red (Type I) muscle fibers.     

Changes in exopeptidase activities in skeletal muscles during disuse

Some aminopeptidase activities, dipeptidase-, tripeptidase-, and carboxypeptidase activities were measured in two different types of skeletal muscle in rabbit soleus muscle as a slow oxidative, and gastrocnemius muscle as a fast glycolytic type after immobilization in full extension with a plaster cast for 1, 2, 4, 7, 14 or 28 days. In correlation to the higher protein turnover in red muscles, the activities except of leucine and alanine aminopeptidase were higher in the normal soleus muscle than in the gastrocnemius muscle. Much higher activities of the tested enzymes were obtained in the immobilized soleus muscle than in the normal one after 2 weeks of immobilization. In the gastrocnemius muscle the tested enzyme activities generally did not change or decrease. The results demonstrate that the peptidases play a role in the process of protein breakdown in normal and disused skeletal muscles.     

Muscle type difference in the regulation of UCP3 under cold conditions

We found opposite regulation of uncoupling protein 3 (UCP3) in slow-twitch soleus and fast-twitch gastrocnemius muscles of rats during cold exposure. Namely, the UCP3 mRNA level was downregulated in the soleus muscles, but upregulated in the gastrocnemius muscles after a 24-h cold exposure. In the analysis of UCP3 protein, we first succeeded in detecting UCP3 short-form as well as the long-form in vivo, which levels were decreased markedly in the cold-exposed soleus muscles. However, the levels of UCP3 and cytochrome oxidase subunit IV were well maintained in the cold-exposed gastrocnemius muscles with a rise in the total mitochondrial protein level, suggesting an increase of total oxidative ability. The fast-twitch muscle rather than the slow-twitch one may play an important role in adaptive responses, including thermogenesis under acute cold exposure.     

beta-Glucuronidase activity in trained red and white skeletal muscle of mice.

We studied the effects of prolonged running exercise (5 days a week, 1.5 h per day at a speed of 17.6 m/min) on the activity of some acid hydrolases (beta-glucuronidase, beta-N-acetylglucosaminidase, acid phosphatase and cathepsin D) and three enzymes of energy metabolism (cytochrome c oxidase, lactate dehydrogenase and creatine kinase) in the distal and in the proximal, the predominantly white and red parts, respectively, of the vastus lateralis-muscle from mice. The acid hydrolase activity levels were 1.24--1.69 higher in untrained red muscle compared to untrained white muscle. The light training applied increased the activity of beta-glucuronidase in both red and white muscle. No other significant training effects were observed in the enzyme activities measured.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: a combined histochemical and biochemical investigation 2. The biochemical investigation and comparison with the histochemical observations.

The reliability of enzyme histochemical semipermeable membrane techniques for the demonstration of acid hydrolases was investigated with a combined histochemical and biochemical study. In part 1 the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings. In m. soleus, m. plantaris, m. gastrocnemius and diaphragm of vitamin E deficient rabbits the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase and beta-glucuronidase is significantly increased. This increase in activity of the investigated acid hydrolases was equal for muscles with an aerobic or an anaerobic metabolism. By means of statistical calculations the activity of the enzymes demonstrated with histochemical techniques was compared with the enzyme activity determined with biochemical techniques. From the results of this investigation it can be concluded that the histochemical semipermeable membrane techniques for the demonstration of activity of acid hydrolases are very reliable. Considering the fact that these techniques are also tissue-saving, they are therefore extremely suitable for the study of catabolic wasting processes in skeletal muscle tissues of patients with inherited or acquired muscular diseases.     

In vitro drug metabolism in male and female athymic,nude mice

Drug metabolism was studied in hepatic microsomal and post microsomal supernatant fractions from male and female athymic nude mice (nu/nu) and heterozygous (+/nu) and homozygous (+/+) wild-type controls. In males, the following enzyme activities were higher in athymic mice than in the wild-type: NADPH cytochrome c reductase, ethylmorphine and aminopyrine N-demethylases, native UDP glucuronyltransferase, and glutathione (GSH) S-aryltransferase. No differences were observed between groups in UDPNAG-activated UDP-glucuronyltransferase, N-acetyltransferase, or aniline hydroxylase activities or in amounts of cytochrome P-450. In female athymic mice, only ethylmorphine and aminopyrine N-demethylase activities were significantly higher than in female wild-type controls (+/+). The female athymic mice had mixed function oxidase activities that were less than the male athymic mice. There were no sex or strain differences in response to treatment with phenobarbital or 3-methylcholanthrene.     

Muscle oxidative capacity during IL-6-dependent cancer cachexia

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.     

EMG-normalised kinase activation during exercise is higher in human gastrocnemius compared to soleus muscle

In mice, certain proteins show a highly confined expression in specific muscle groups. Also, resting and exercise/contraction-induced phosphorylation responses are higher in rat skeletal muscle with low mitochondrial content compared to muscles with high mitochondrial content, possibly related to differential reactive oxygen species (ROS)-scavenging ability or resting glycogen content. To evaluate these parameters in humans, biopsies from soleus, gastrocnemius and vastus lateralis muscles were taken before and after a 45 min inclined (15%) walking exercise bout at 69% VO2(max) aimed at simultaneously activating soleus and gastrocnemius in a comparable dynamic work-pattern. Hexokinase II and GLUT4 were 46-59% and 26-38% higher (p<0.05) in soleus compared to the two other muscles. The type I muscle fiber percentage was highest in soleus and lowest in vastus lateralis. No differences were found in protein expression of signalling proteins (AMPK subunits, eEF2, ERK1/2, TBC1D1 and 4), mitochondrial markers (F1 ATPase and COX1) or ROS-handling enzymes (SOD2 and catalase). Gastrocnemius was less active than soleus measured as EMG signal and glycogen use yet gastrocnemius displayed larger increases than soleus in phosphorylation of AMPK Thr172, eEF2 Thr56 and ERK 1/2 Thr202/Tyr204 when normalised to the mean relative EMG-signal. In conclusion, proteins with muscle-group restricted expression in mice do not show this pattern in human lower extremity muscle groups. Nonetheless the phosphorylation-response is greater for a number of kinase signalling pathways in human gastrocnemius than soleus at a given activation-intensity. This may be due to the combined subtle effects of a higher type I muscle fiber content and higher training status in soleus compared to gastrocnemius muscle.     

Developmental Changes in Lactate Dehydrogenase and Aldolase Activity of the A2G-adr Mouse with Abnormal Muscle Function: Further Comparison with the 129Re-dy Mutant

Abstract: Lactate dehydrogenase and aldolase activity were reduced in lateral gastrocnemius muscle from two mouse mutants, A2G- adr and 129Re- dy , with abnormal muscle function. The activities of both of these enzymes were significantly reduced in the lateral gastrocnemius muscle from the A2G- adr mice at ages varying from 2 weeks to 32 weeks, whereas the activities in the soleus, heart, liver, and brain were the same as in the control animals. The lactate dehydrogenase isoenzymes in the lateral gastrocnemius and soleus muscles from the A2G mice were quantified, and although those of the soleus were comparable in mutant and control muscle, the lateral gastrocnemius from the adr mutant had reduced activity of LDH 5 and increased activities of the other four isoenzymes. The findings suggest that the adr mutation is expressed in the white (Type II) muscle fibres and not in the red (Type I) fibres or in any of the organs studied. It is suggested that the initiation of differentiation into Type II fibres from the embryonic form is absent or delayed in the A2G mutant. The reduced activities of lactate dehydrogenase and aldolase in 129Re- dy muscle confirm the findings of other workers.     

Metformin increases the PGC-1alpha protein and oxidative enzyme activities possibly via AMPK phosphorylation in skeletal muscle in vivo.

AMP-activated protein kinase (AMPK), which was activated by an antihyperglycemic drug metformin, has been hypothesized to mediate metabolic adaptations. The purposes of the present study were 1) to confirm whether acute metformin administration induced AMPK phosphorylation and 2) to determine whether chronic metformin treatment increased the peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) protein expression, glycolytic and oxidative enzyme activities, and cytochrome c and glucose transporter-4 (GLUT4) protein expressions in the rat soleus and red and white gastrocnemius muscles. The single oral administration of metformin (300 mg/kg body wt) enhanced the AMPK phosphorylation at 5 and/or 6 h after treatment. In the chronic study, rats were fed either normal chow or chow containing 1% metformin for 14 days. Metformin treatment resulted in a mean daily metformin intake of 631 mg.kg body wt(-1).day(-1). Metformin increased the PGC-1alpha content in all three muscles. Metformin increased the hexokinase activity in the white gastrocnemius, the citrate synthase activity in all three muscles, and the beta-hydroxyacyl-CoA dehydrogenase activity in the soleus. The cytochrome c protein content in the soleus muscle also increased. The GLUT4 content was unchanged by metformin. These results suggest that metformin enhances the PGC-1alpha expression and mitochondrial biogenesis possibly at least in part via AMPK phosphorylation in the skeletal muscle. Metformin has thus been proposed to possibly ameliorate insulin resistance, at least partially, by means of such metabolic effects.     

Temperature adaptation of biological membranes. Compensation of the molar activity of cytochrome c oxidase in the mitochondrial energy-transducing membrane during thermal acclimation of the carp (Cyprinus carpio L.)

The acclimation temperature of carp does not affect the amount of cytochrome c oxidase per mg mitochondrial protein as revealed from the reduced-minus-oxidized difference spectra of red muscle mitochondria from cold- and warm-acclimated carp. There are no differences between cold- and warm-acclimated fish in the substrate binding properties of the enzyme as judged from the Km values for cytochrome c at 30 degrees C (3.34 +/- 0.ee microM, acclimation temperature 10 degrees C and 3.55 +/- 0.31 microM, acclimation temperature 30 degrees C). The molar activities of the enzyme, however, differ for both acclimation temperatures: when intercalated in the 10 degrees C-acclimated mitochondrial membrane, the enzyme can catalyze the oxidation of 117.6 +/- 17.2 mol ferrocytochrome c/s per mol heme a as compared with 85.6 +/- 17.2 in the 30 degrees C-acclimated membrane (experimental temperature 30 degrees C). Correspondingly, higher specific activities of the succinate oxidase system are observed in mitochondria from cold-acclimated carp as compared with those obtained from warm-acclimated carp. The results indicate that cold acclimation of the eurythermic carp is accompanied by a partial compensation of the acute effect of decreasing temperature on the activity of cytochrome c oxidase in red muscle mitochondria. Based on the temperature-induced lipid adaptation reported for carp red muscle mitochondria (Wodtke, E. (1980) Biochim. Biophys. Acta 640, 698--709), it is concluded that during thermal acclimation the molar activity of cytochrome c oxidase is controlled by viscotropic regulation. The results fit to the conception that cardiolipin constitutes a lipid shell (annulus) surrounding the oxidase within the native membrane, but that it is the bilayer fluidity and not the annular fluidity which determines the activity of cytochrome c oxidase.     

Comparisons of copper deficiency states in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice.

The activity of two copper-dependent enzymes, cytochrome c oxidase and copper, zinc-superoxide dismutase, was determined in six tissues of age-matched (13-day-old) copper-deficient mutant and normal mice. In the two mutants 'brindled' and 'blotchy', brain, heart and skeletal muscle had significant enzyme deficiencies. Cytochrome c oxidase was more severely affected than was superoxide dismutase. In these three tissues the degree of deficiency could be correlated with decreased copper concentration; however, enzyme activity was normal in liver, kidney and lung, despite abnormal copper concentrations in these tissues. In nutritionally copper-deficient mice, all six tissues showed decreased enzyme activity, which was most marked in brain, heart and skeletal muscle, the tissues which showed enzyme deficiencies in the mutants. Analysis in vitro of cytochrome c oxidase (temperature coefficient = 2) at a single temperature was found to underestimate the deficiency of this enzyme in hypothermic copper-deficient animals. Cytochrome c oxidase deficiency may therefore be sufficiently severe in vivo to account for the clinical manifestations of copper deficiency. An injection of copper (50 micrograms of Cu+) at 7 days increased cytochrome c oxidase activity by 13 days in all deficient tissues of brindled mice, and in brain and heart from blotchy mice. However, skeletal-muscle cytochrome c oxidase in blotchy mutants did not respond to copper injection. Cytochrome c oxidase activity increased to normal in all tissues of nutritionally copper-deficient mice after copper injection, except in the liver. Hepatic enzyme activity remained severely deficient despite a liver copper concentration three times that found in copper-replete controls. Superoxide dismutase activity did not increase with treatment in either mutant, but its activity was higher than control levels in nutritionally deficient mice after injection. This difference is probably due to sequestration of copper in mutant tissue such as kidney, but a defect in the copper transport pathway to superoxide dismutase cannot be excluded.     

Endurance training decreases the alkaline proteolytic activity in mouse skeletal muscles

Alkaline and myofibrillar protease activities of rectus femoris, soleus, and tibialis anterior muscles and the pooled sample of gastrocnemius and plantaris muscles were analyzed in male NMRI-mice during a running-training program of 3, 10, or 20 daily 1-h sessions. The activity of citrate synthase increased during the endurance training, reflecting the increased oxidative capacity of skeletal muscles. The activities of alkaline and myofibrillar proteases continually decreased in the course of the training program in all muscles studied. Instead, the activity of beta-glucuronidase (a marker of lysosomal hydrolases) increased in all muscles. The highest activities were observed at the beginning of the training program. Present results, together with our earlier observations, show that the type of training, running as opposed to swimming, modulates the training responses in alkaline protease activities. Further, diverse adaptations in the activities of alkaline proteases and a lysosomal hydrolase suggest difference in the function of different proteolytic systems.     

Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice

Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Mo(vbr) (mottled viable brindled) mice, an animal model of Menkes' copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Mo(vbr) mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Mo(vbr) and control mice. Inhibitor titrations were used to determine flux control coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar flux control coefficients for control and mutant animals. Alternately, titration of respiration with azide revealed for Mo(vbr) mice significantly sharper inhibition curves than for controls, indicating a more than twofold elevated flux control coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR.