Metabolic profile and nitric oxide synthase expression of skeletal muscle fibers are altered in patients with type 1 diabetes.

We investigate muscle fiber composition, fiber-specific glycolytic and oxidative enzyme capacity and nitric oxide synthase (NOS) expression in skeletal muscle of patients with type 1 diabetes (T1D) compared to individuals with normal glucose tolerance (NGT). Vastus lateralis muscle was obtained by percutaneous biopsy from 7 T1D patients and 10 healthy controls with similar characteristics. Using cytophotometry, muscle fiber composition and fiber type-specific glycolytic and oxidative enzyme activities were measured in slow oxidative (SO), fast oxidative glycolytic (FOG) and fast glycolytic (FG) fibers. In addition, NOS 1-3 protein expression was mea-sured. The glycolytic fiber fraction was 1.4 fold higher, whereas FOG and SO fiber fractions were significantly reduced by 13.5% and 6.2% in skeletal muscle from T1D patients. Glycolytic enzyme activities and fiber-specific ratio of glycolytic relative to oxidative enzyme activity were significantly higher in all fiber types of T1D patients and correlated with HbA (1c). Expression of NOS1-3 isoforms was reduced in skeletal muscle of T1D subjects. Increased glycolytic enzyme activity in muscle of T1D patients is most likely due to both a higher number of fast glycolytic fibers and a shift towards increased glycolytic metabolism in all fiber types. Alterations in muscle fiber distribution and enzyme activities seem to be due to impaired long-term glycemic control.

Fibre-related nitric oxide synthase (NOS) in Duchenne muscular dystrophy.

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The loss of NO synthase (NOS) from the sarcolemma was assumed to be associated with development of Duchenne muscular dystrophy (DMD). We have, however, recently reported that, in contrast to the commonly accepted view, NOS expression in DMD myofibres is up-regulated. This poses the question of the fibre type-specific NOS expression in DMD muscles and how the NOS expression is related to the regeneration or degeneration status. To address this issue, we examined localization of NOS isoforms I, II and III in skeletal muscles of DMD patients employing immunohistochemical labelling with tyramide signal amplification complemented with enzyme histochemistry. We found that NOS immunolabelling as well as metabolic enzyme activity in DMD muscles were heterogeneously distributed along the fibre length of DMD muscle fibres revealing regenerating and degenerate (hypercontracted) fibres as well as normal segments. Like in normal muscles, positive NOS immunoreactivity was found to be associated with fast-oxidative glycolytic (FOG) phenotype. The regeneration status of NOS-positive segments was deduced from the presence of neonatal and developmental myosin heavy chains. High NOS expression in regenerating DMD muscle fibres can be well reconciled with reports about the protective role of endogenous NO in inflammatory diseases and in muscle repair.

Nitric oxide synthase isoforms I, III and protein kinase-Ctheta in skeletal muscle fibres of normal and streptozotocin-induced diabetic rats with and without Ginkgo biloba extract treatment.

The expression of nitric oxide synthase (NOS) isoforms I, III and protein kinase-Ctheta (PKCtheta) in rat vastus lateralis muscle was demonstrated immunohistochemically and then correlated to the physiological metabolic fibre types: SO (slow-oxidative), FOGI, FOGII (fast-oxidative glycolytic; I more glycolytic, II more oxidative), and FG (fast-glycolytic). NOS expression in muscles from different experimental groups (normal and diabetic rats, with and without Ginkgo biloba extract treatment) was assayed by Western blotting. Generally, NOS I and PKCtheta were co-expressed in fibres with predominantly oxidative metabolism (SO, FOGII). This suggests an interplay of PKCtheta and NOS I in nitric oxide production by oxidative fibres. NOS III was more highly expressed in fibres with predominantly glycolytic metabolism (FOGI, FG). A somewhat lower NOS I immunoreactivity was also found in NOS III positive fibres suggesting that NOS III and NOS I are co-expressed in these fibres. Western blotting revealed that NOS I as well as NOS III expression in the vastus lateralis muscle was down-regulated in diabetes and increased after Ginkgo biloba extract treatment. These effects may be associated with a diminished glucose uptake by myocytes of diabetic musclesand with an improved muscle function after Ginkgo biloba treatment.

Changes of enzyme activities in the myocardium and skeletal muscle fibres of cardiomyopathic hamsters. A cytophotometrical study.

Cytophotometrical measurements of enzyme activities were performed in the myocardium and skeletal muscle fibres from normal and cardiomyopathic hamsters (BIO 8262) during ageing from 12-14 to 120-190 days. Myocardium as well as vastus lateralis muscles of cardiomyopathic hamsters showed changes in enzyme activities. The skeletal muscle fibres were typed into slow-oxidative, fast-oxidative glycolytic and fast-glycolytic to investigate fibre type-related changes in muscles of cardiomyopathic hamsters. The following myopathic changes were mainly found: Myofibrillic ATPase was depressed in the myocardium of both ventricles in all investigated age stages. The ATPase activity of the right ventricle was more decreased than that of the left one. Additionally, a metabolic shift was observed in myocardium and slow-oxidative muscle fibres at the onset of clinical symptoms, which appeared from day 150 to day 190. During the period from 42 up to 190 days of life an increase of oxidative (succinate dehydrogenase) activity was measured in the myocardium of both ventricles and in slow oxidative fibres of vastus lateralis muscle as a proximal muscle. At earlier ages, the fast fibres of myopathic vastus lateralis muscle showed higher glycolytic (glycerol-3-phosphate dehydrogenase) activity than those of normal muscles. However, at the age of 120-190 days the metabolic profile of fast fibres was normalized. In gastrocnemius muscle as a distal muscle no changes of enzyme activities were measured, suggesting the investigated hereditary myopathy effected proximal, but not distal muscles.

Effects on skeletal muscle fibres of diabetes and Ginkgo biloba extract treatment.

Combined cytophotometric and morphometric analysis of muscle fibre properties and myosin heavy chain electrophoresis were performed on extensor digitorum longus and soleus muscles from healthy rats and rats with streptozotocin-induced diabetes. Moreover, the protective effect of Ginkgo biloba extract, a potent oxygen radical scavenger, on diabetic muscles was investigated. Changes in fibre type-related enzyme activities, fibre type distribution, fibre cross areas and myosin isoforms were found. In muscles of diabetic rats, a metabolic shift was measured mainly in fibres with oxidative metabolism. Fast-oxidative glycolytic fibres showed a shift to more glycolytic metabolism and about a third transformed into fast-glycolytic fibres. Slow-oxidative fibres became more oxidative. Fibre atrophy was measured in diabetic muscles dependent on fibre type and muscle. Different fibre types atrophied to a different degree. Therefore, a decreased area percentage of slow fibres and an increased area percentage of fast fibres of the whole muscle cross section in both muscles were found. This is supported by reduced slow and increased fast myosin heavy chain isoforms. These alterations of diabetic muscle fibres could be due to less motion of diabetic rats and diabetic neuropathy. After treatment with Ginkgo biloba extract, enzyme activities were increased mainly in oxidative fibres of diabetic muscles, which was interpreted as protective effect. Generally, the soleus muscle with predominant oxidative metabolism was more vulnerable to diabetic alterations and Ginkgo biloba extract treatment than the extensor digitorum longus muscle with predominant glycolytic metabolism.

Expression of BCL-2 in the developing kidney of human embryos and fetuses qualitative and quantitative study.

Twelve human embryos and fetuses aged of 7-30 weeks of intrauterinal life were examined to determine the expression of bcl-2 gene in the developing kidney. Tissue samples were routinely processed and three-step indirect immunohistochemical method was used for the detection of Bcl-2 protein. End-point cytophotometry was performed with computer-controlled microscope photometer with a scanning table and the mean relative absorbance of the final product of peroxidase reaction was determined and taken as a measure of Bcl-2 expression. The morphometric evaluation was carried out from the TV display using Weibel s universal hexagonal raster and we determined the relative volume of Bcl-2 positive structures in the various zones of the embryonal kidney. The aim of our research was mapping of the Bcl-2 occurrence in the developing kidney of human embryos and fetuses. The Bcl-2 protein is involved in the regulation of apoptosis and its effect is antiapoptotic. The highest Bcl-2 expression was proved in the cells of metanephrogenic blastema. The lower occurrence of Bcl-2 positive cells was demonstrated in proximal tubules analges+ and it was almost on the borderline of detection in branches of ureteral bud. In the fetal period the marked Bcl-2 expression was maintained in the epithelial cells of proximal tubules analges.

Region- and age-dependent variations of muscle fibre properties.

The cytophotometric-morphometrical analysis of extensor digitorum longus and soleus muscles of 2.5 and 18 months old rats revealed regional and age-dependent differences in fibre type distribution, fibre area and fibre type related-enzyme activities which characterize contractility and metabolic profile. Variations along the longitudinal axis from the origin to the insertion and along three transversal axes from superficial to deep were found dependent on the muscle investigated. For example, the fibres of extensor digitorum longus muscle showed increased contractile and glycolytic capacities near insertion and the fibres of soleus muscle increased oxidative capacity in its middle part. Furthermore, the contribution of the fibre type that is dominant in a muscle (fast-glycolytic fibre type in extensor digitorum longus and slow-oxidative fibre type in soleus muscle) to the total number of fibres increased from origin to insertion by 15 and 30%, respectively. Along the superficial-deep axes the oxidative capacity of all fibres increased, the most in fast fibres of the soleus muscle by approximately 50%. In soleus muscle, a decrease of cross areas of all fibre types from superficial to deep was found, correlating negatively with the succinate dehydrogenase activity of the fibres. In extensor digitorum longus muscle the change in cross areas of slow-oxidative and fast-oxidative glycolytic fibres was dependent on the position of the transversal axis in the muscle. The results suggest that distribution patterns of fibre types and the metabolic make up of individual muscle fibres are adapted on the basis of local functional demands. In both muscles, higher numbers and increased oxidative capacity of fast-glycolytic fibres were found during ageing, but variations from superficial to deeper regions were irrespective of age.

The correlation of cytophotometrically and biochemically measured enzyme activities: changes in the myocardium of diabetic and hypoxic diabetic rats, with and without Ginkgo biloba extract treatment.

Changes of enzyme activities in the myocardium of rats from 6 different experimental groups (normal rats, diabetic rats, hypoxic diabetic rats, each with and without Ginkgo biloba extract treatment) were measured by using both cytophotometric and biochemical methods. The activity of succinate dehydrogenase, a marker of oxidative capacity, and of menadione-dependent glycerol-3-phosphate dehydrogenase and total lactate dehydrogenase, both markers of glycolytic capacity were measured to characterize changes of the metabolic profile in myocardium. A strong correlation between cytophotometric and biochemical data were found by linear regression analysis, justifying the use of cytophotometrical enzyme activity measurements in cells of organized tissue, where biochemistry cannot provide topographical information. The comparison of the results obtained from the different groups revealed the following: Enzyme activities in the myocardium of rats with streptozotocin-induced diabetes were significantly increased by 10-30% as compared to the normal myocardium. This effect was interpreted as a metabolic compensation of the diabetic heart with reduced performance. When diabetic rats were exposed to acute hypoxia of 20 min duration, enzyme activities decreased under the normal level, to 56% of the succinate dehydrogenase activity, to 87% of glycerol-3-phosphate dehydrogenase activity and to 69% of lactate dehydrogenase activity. Treatment of rats with the oxygen radical scavenger Ginkgo biloba extract (EGb 761) over 3 months resulted primarily in an increase by 10% of oxidative capacity and in a decrease by 30% of glycolytic capacity. Under diabetic conditions a shift to more glycolytic metabolism was observed by increasing the glycolytic activity by 39% and remaining the oxidative activity.

24-hour rhythmicity of NADPH-diaphorase activity in the neuropil of rat visual cortex.

In the present study we examined the daytime-dependent alterations of nitric oxide synthase in the visual cortex of the rat. For this purpose, the activity of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d), an enzyme equivalent to nitric oxide synthase, was measured histochemically in rat visual cortex at 0600, 1200, 1800, and 2400 h using a photometric scanning method. Our results show day-time-dependent changes of the NADPH-d activity in the neuropil of the visual cortex. This was highest at 0600 h and decreased between 1200 h and 1800 h (unimodal profile of circadian activity). The number of NADPH-d-positive neuronal somata was not found to vary at the different time points.

Hypoxia-dependent changes of enzyme activities in different fibre types of rat soleus and extensor digitorum longus muscles. A cytophotometrical study.

Using cytophotometry activity changes of succinate dehydrogenase, glycerol-3-phosphate dehydrogenase and myofibrillar adenosine triphosphatase were measured in 3 fibre types of soleus and extensor digitorum longus muscles under normal and experimental conditions. Fibres were typed by means of cytophotometrical data into slow-oxidative, fast-oxidative glycolytic and fast-glycolytic ones. After experimental hypoxia of 20 min duration a significant increase of enzyme activities was observed especially in slow-oxidative and fast-oxidative glycolytic fibres of both muscles, e.g. succinate dehydrogenase activity increased by 21% in these fibres of soleus muscle and by 23-26% in these fibres of extensor digitorum longus muscle. Moreover, an increase of glycerol-3-phosphate dehydrogenase activity by 10% in slow-oxidative fibres and by 28% in fast-oxidative glycolytic fibres and a 10-12% increased ATPase activity in all fibres of extensor digitorum longus muscle were measured. Treatment with Ginkgo biloba extract for 3 months before exposure to hypoxia resulted in increased adenosine triphosphatase activity in all fibres of both muscles and in decreased succinate dehydrogenase activity of slow-oxidative and fast-oxidative glycolytic fibres of extensor digitorum longus muscle. These results could be interpreted as a protective effect of Ginkgo biloba extract.

Enzyme histochemistry in fetal human hearts and kidneys during intrauterine development. Qualitative and quantitative results.

The activities of three enzymes, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and dipeptidylpeptidase IV, were histochemically demonstrated and cytophotometrically measured in fetal human hearts and kidneys. During the intrauterine development from the 7th to the 19th week 29 fetuses in 6 stages of age were investigated. Positive enzyme reaction was found in the myocardium of left and right ventricle and septum and in the different parts of the nephron. During the investigated period of development the enzyme activities changed in both heart and kidney. At the 16th week the activities of glycerol-3-phosphate dehydrogenase and succinate dehydrogenase showed the maximum in the heart, in contrast to kidney, where at this stage activity minima of succinate dehydrogenase and dipeptidylpeptidase IV were measured. The myocardium of the right ventricle showed higher metabolic activity than the right one, which could reflect the higher load of the right ventricle in the fetal circulation. Within the nephron differences of enzyme activities were observed. The highest enzyme activities were always measured in the proximal tubules, interpreted as sign of high degree of differentiation.

Changes of enzyme activities in the rat myocardium caused by experimental hypoxia with and without ginkgo biloba extract EGb 761 pretreatment. A cytophotometrical study.

Using cytophotometry activity changes of succinate dehydrogenase, glycerol-3-phosphate dehydrogenase and myofibrillar adenosine triphosphatase, were measured in the rat myocardium under normal and different experimental conditions. After hypoxia all enzyme activities were significantly decreased in comparison to the normal situation, and the alterations differed in both ventricles. Ginkgo biloba extract treatment over three months before exposition to hypoxia resulted in a lower inhibition of succinate dehydrogenase, a higher inhibition of glycerol-3-phosphate dehydrogenase and an unchanged activity of adenosine triphosphatase after hypoxia of 20 min. These results were interpreted as a protective effect of the Ginkgo biloba extract on the hypoxic myocardium.

Enzyme activity patterns of myosin ATPase, alpha-glycerophosphate dehydrogenase and succinate dehydrogenase within different muscle fibre types.

Muscle fibre compositions of five different rabbit muscles were determined by combining two enzyme-histochemical reactions (NADH tetracolium oxidoreductase and myosin ATPase after alkaline preincubation). The differentiation into the fibre types, fast twitch glycolytic (FTG), fast twitch oxidative (FTO), and slow twitch oxidative (STO) was possible by a reliable staining classification. Aim of the study was the estimation of enzyme activity patterns within the three different fibre types. For this purpose, four serial cross-sections with several enzyme histochemical reactions were performed: alkaline combination method for fibre type determination, the reactions of myosin ATPase, alpha-glycerophosphate dehydrogenase (GPDH), and succinate dehydrogenase (SDH). The measurement procedure for the estimation of enzyme activities was based on the proportionality between the intensity of the enzyme histochemical staining reaction and the degree of enzyme activity. The activities of GPDH (indicator for glycolytic metabolism) and SDH (oxidative metabolism) were inverse. The calcium-activated myosin ATPase showed only little activity in slow twitch fibres after alkaline preincubation. In contrast to slow twitch fibres, ATPase activity in fast twitch fibres was relatively high. The results showed that the classification of muscle fibre types due to their myosin ATPase activities and their main metabolisms (oxidative and glycolytic respectively) is justified.

Age-dependent changes of enzyme activities in the different fibre types of rat extensor digitorum longus and gastrocnemius muscles.

The age-dependent change of metabolic profiles of SO (slow-oxidative), FOG (fast-oxidative glycolytic) and FG (fast-glycolytic) fibres of muscles digitorum longus and musculus gastrocnemius of rat from 14 days to 370 days was measured cytophotometrically. Fibres were classified visually and using cytophotometrical data from staining reactions for myofibrillar adenosinetriphosphatase (ATPase), succinate dehydrogenase (SDH) and alpha-glycerophosphate dehydrogenase (alpha-GPDH) in the same fibre. The fibre type population as percentage was estimated at different ages. The age-dependent change of enzyme activities was demonstrated in each fibre type. SDH-heterogeneity of FOG-fibres and consequently an overlap with SO-fibres was detected. The alpha-GPDH/SDH-activity quotient allowed to distinguish SO-, FOG- and FG-fibres, and the age-dependent change of activity quotient characterized the change of metabolic properties in the concerned fibre types. Whereas in gastrocnemius muscle the metabolic profile of FOG-fibres was similar to that of SO-fibres, in extensor digitorum longus muscle the metabolism of FOG-fibres was similar to that of FG-fibres. Between the two muscles differences were also shown for the fibre type responsible for changes of enzyme activities in the whole muscle, measured biochemically.

Histophotometry--the method of choice in quantifying dehydrogenase histochemistry.

Succinate, malate, and lactate dehydrogenase were demonstrated histochemically and measured histophotometrically in the heart and skeletal muscle (m. extensor digitorum longus and m. soleus) of rats at different ages. To prove the value of histophotometry, the enzymes of the tissues were estimated biochemically. The gel film technique cannot sufficiently prevent the diffusion of the soluble enzymes (malate-, lactate dehydrogenase) out of the tissue sections. Because of the different mobility, various isoenzymes, histophotometry cannot give reliable results. But, as far as membrane-bound dehydrogenases (succinate dehydrogenase) are concerned, histophotometry is the method of choice for basic measurements as in routine practical work, especially with tissues where the enzyme activities are heterogeneously distributed, e.g. in different types of muscle fibres in skeletal muscles.

Ultramicroscopic morphometric, biochemical and histophotometric investigations of myocardial biopsies taken from Fallot patients before and after cardioplegia with cold Kirsch's solution.

Biopsies taken from the myocardium of 5 patients with Morbus Fallot and from 1 patient with ventricle septum defect were investigated with a combination of morphometric, biochemical and histophotometric techniques in order to study the cardioprotective effect of cold Kirsch's solution. At the final phase of cardioplegia the cardiomyocytes reveal the following alterations: The volume densities of mitochondria, of their degenerated areas and that of cytoplasmic vacuoles show a significant increase whereas that of myofilaments decreases. Cristae and matrix mitochondriales, however, show only moderate alterations without statistical significance. Biochemically the total ATP-concentration and creatine phosphate (CP)-concentration were more or less diminished, in most of the cases the activity of the myosin ATPase was increased, that of the creatine phosphate kinase (CPK) diminished. Compared with the biochemical estimations of the ATPase activity, its histophotometric estimations yielded corresponding results in 2 of 4 cases. In general our findings confirm the cardioprotective effect of Kirsch's solution. The combination of methods used gives more reliable results than one technique alone.

Histophotometrical measurements concerning the distribution of myofibrillar ATPase activity within the tissue block after aldehyde fixation.

The inhibition of myofibrillar ATPase activity by aldehyde fixation in tissue pieces of myocardium of the rat was measured by scanning histophotometry. The relative amount of the final reaction product of the histochemical method for ATPase (Padykula and Herman 1955) was taken as a measure for the enzyme activity. Along scanning lines in slides from the surface to the centre of tissue block, a higher activity was found in the centre in contrast to the marginal zone. Compared with the centre of the block (approximately equal to 100%), in the marginal region a loss of about 40% (in paraformaldehyde PFA) and of 75% (in glutaraldehyde GA) was found, the latter with a sharp decline between the marginal zone and the central part. For quantitative enzyme histochemistry by histophotometry unfixed material is recommended.

The higher myosin ATPase activity in the right heart ventricle of the rat, proved by histophotometry.

Comparing the myosin-ATPase activity in the left and right ventricular wall of the hearts from 6 rats by scanning histophotometry, a higher enzyme activity in the right ventricle was found. These findings are in a good agreement with the higher numbers of myofilaments per myofibril in the right ventricle, reported in literature.

The correlation between histophotometrical and biochemical myosin-ATPase measurements in the myocardium and striated muscle of the rat.

To prove the correlation between histophotometrical and biochemical data of myosin-ATPase-activity, the change of enzyme activity during development was measured in the myocardium, masseter muscle, and tensor fasciae latae of rats. Both methods were applied to the same material. To avoid errors due to varying section thickness in each cutting and staining procedure, a reference material (e.g. heart muscle) was used, taken from one and the same animal. The correlation between histophotometrical and biochemical estimations was very good, histophotometrical arbitrary units being used. ATPase-activity in heart muscle during development, slightly decreases in masseter muscle and more in tensor fasciae latae. The ATPase-activity in the latter was higher than in masseter muscle. This is in good agreement with the physiological, biochemical, and histochemical classification.

Localization artefacts in ultracytochemical ion precipitation reactions.

The precipitation patterns of the following ultracytochemical methods in rat muscle cells were compared and examined critically: the potassium pyroantimonate method for calcium demonstration; the calcium phosphate technique for the Ca2+--ATPase reaction; the formazan reaction for the demonstration of creatine kinase activity (all performed on heart muscle); and the lead phosphate technique for the Mg2+--ATPase reaction in skeletal muscle. Using X-ray microanalysis, it was found that the antimonate precipitate contains only calcium as the precipitated ion in the vast majority of cases. Most probably it consists of pure calcium pyroantimonate. However, in myocytes showing the well-established precipitation pattern, the concentration of calcium was estimated to be about two orders of magnitude higher than the native concentration of total intracellular calcium. It is concluded that calcium ions diffuse freely from the extracellular space and from adjacent cells into cells containing antimonate and are precipitated mostly at sites where heterogeneous nucleation is facilitated by intracellular catalysts (biopolymers). As shown by the similar precipitation patterns for the four reactions compared, these catalysts are not specific to any of these reactions and are most probably neither calcium-binding sites nor sites of any one of the enzymes examined in the native cell.