GLYCOGEN AND GLYCOGEN PHOSPHORYLASE IN THE CEREBRAL CORTEX OF MICE UNDER THE INFLUENCE OF METHIONINE SULPHOXIMINE

Abstract— Glucose and glycogen levels in the mouse cerebral cortex in vivo were studied after recovery from methionine sulphoximine seizures. The animals appeared normal 24 h after methionine sulphoximine administration but both glucose and glycogen still persisted at higher levels 72 h after injection (by 64 and 275 per cent, respectively). When seizures were prevented by methionine, the increase in glucose and glycogen at the longer time intervals was significantly smaller than in animals treated with methionine sulphoximine only; glucose reached normal values at 48 or 72 h; the accumulation of glycogen was reduced by about three to five times, but after 72 h the levels were still significantly higher than in control animals (67 or 32 per cent increase, depending on the administered dose of methionine). In contrast to the considerable accumulation of glycogen after administration of methionine sulphoximine in vivo, it had no effect on the level of glycogen in brain cortex slices in vitro. After 3 h incubation in the absence of methionine sulphoximine, glycogen was resynthesized to a level of about 4 μmol/g wet tissue and this value was not significantly affected by the presence of various concentrations of methionine sulphoximine in the incubation medium (10^-5 to 10^-2 M). The total (a+b forms) phosphorylase activity of mouse cerebral cortex in vivo after methionine sulphoximine administration was not affected. The fraction of active phosphorylase was reduced by about 50 per cent at the time of seizures. When seizures were prevented by methionine, the decrease in active phosphorylase was also completely prevented. In the preconvulsive period (1-2 h) and after recovery from the seizures (48 h after methionine sulphoximine administration) active phosphorylase was normal. The possible mechanisms involved in the increased accumulation of glycogen after methionine sulphoximine administration are discussed.     

CHANGES IN GLYCOGEN PHOSPHORYLASE ACTIVITY AND GLYCOGEN LEVELS OF MOUSE CEREBRAL CORTEX DURING CONVULSIONS INDUCED BY HOMOCYSTEINE

Glycogen phosphorylase activity and glycogen levels were investigated in the cerebral cortex of mice of two different strains under the influence of homocysteine. Control levels of glycogen and total phosphorylase activity (i. e. activity in the presence of 1 mM-AMP) were higher in the inbred strain A, whereas a higher proportion of phosphorylase in its active form (activity without 5′-AMP) was obtained in the ICR strain (probably due to slower fixation of brain in this strain). Changes occurring after the administration of homocysteine were similar in both strains. With the onset of first clonic seizures a marked increase of phosphorylase a occurred (increase 99 per cent in strain A and 46.5 per cent in ICR, respectively). During the latter phase of tonic seizures active phosphorylase a did not significantly differ from control values. Five minutes after the end of a tonic seizure, i. e. when partial recovery could already be observed, a marked decrease of active phosphorylase a in comparison with control values, was evident (decrease against control values of 45.5 per cent in strain A and 30.5 per cent in ICR, respectively). The total phosphorylase activity was not affected in strain A, whereas a slight increase during clonic seizures was seen in the ICR strain. In accordance with the enhanced activation of phosphorylase at the onset of clonic seizures, a marked decrease in glycogen levels (35-50 per cent) was observed in both strains of mice. This decrease persisted even during the 5 min recovery period. When seizures were prevented by Na phenobarbital or glycine, the activation of phosphorylase was either completely prevented (by a non-anaesthetic dose of phenobarbital) or reduced (by glycine). The present results have demonstrated that changes in glycogen metabolism occurring during homocysteine seizures differ distinctly from those previously found during seizures induced by methionine sulphoximine, a substance structurally related to homocysteine.     

SOME FACTORS INFLUENCING BRAIN GLYCOGEN IN THE NEONATE CHICK

—Studies of the brain glycogen concentration in the chick during the perinatal period showed that there was an increase immediately prior to hatching. This was followed by a pronounced decrease between 1 and 2 days after hatching. The decrease was most marked in the cerebellum. During ischaemia, the rate of glycogen depletion was greater in 1-day-old chicks than in 2- and 7-day-old birds. Brain glycogen concentration exhibited a circadian rhythm which was not closely related to changes in motor activity or body temperature. Exposure to a high environmental temperature (40°C) caused a depletion of glycogen, but exposure to a low temperature (2°C) had no effect. Four hours of hyperglycaemia resulted in a lowering of brain glycogen levels whereas hypoglycaemia was without effect.     

IDENTIFICATION OF GLYCOGEN IN ELECTRON MICROGRAPHS OF THIN TISSUE SECTIONS

The electron microscopic appearance of glycogen has been studied in the organs of several animal species. Glycogen almost always appears as roughly circular granules from 150 to 400 A in diameter. The intrinsic electron density of glycogen varies from tissue to tissue; however, treatment with lead hydroxide as described by Watson deeply stains the granules. Glycogen pellets were isolated from some of the tissues studied by centrifugation. Such pellets were shown to be glycogen by chemical and histochemical criteria. When thin sections of the pellet are examined under the electron microscope they can be seen to consist of densely packed granules similar to those found in the intact tissues. Such pellets are also stained for electron microscopy by short exposure to lead hydroxide.     

海洋氢弧菌胞内糖原生理功能的研究

在细胞高密度培养后的各种不同条件下,通过停止提供碳源和洗涤细胞培养法来观察专性化能自养海洋氢弧菌（Hydrogenovibriomarinus）胞内糖原和胞内葡萄糖碳酸酶活性变化发现：这株自养细菌胞内的糖原起能量储存的作用。最大的糖原降解为76．5％,是发生在碳源和能源饥饿的有氧状态下。     

AUTOPHAGIC DEGRADATION OF GLYCOGEN IN SKELETAL MUSCLES OF THE NEWBORN RAT

Large amounts of glycogen accumulate in rat skeletal muscle fibers during the late fetal stages and are mobilized in the first postnatal days. This glycogen depletion is relatively slow in the immature leg muscles, in which extensive deposits are still found 24 hr after birth and, to some extent, persist until the 3rd day. In the more differentiated psoas muscle and especially in the diaphragm, the glycogen stores are completely mobilized already during the early hours. Section of the sciatic nerve 3 days before birth or within the first 2 hr after delivery does not affect glycogen depletion in the leg muscles. Neonatal glycogenolysis in rat muscle fibers takes place largely by segregation and digestion of glycogen particles in autophagic vacuoles. These vacuoles: (a) are not seen in fetal muscle fibers or at later postnatal stages, but appear concomitantly with the process of glycogen depletion and disappear shortly afterwards; (b) are prematurely formed in skeletal muscles of fetuses at term treated with glucagon; (c) contain almost exclusively glycogen particles and no other recognizable cell constituents; (d) have a double or, more often, single limiting membrane and originate apparently from flattened sacs sequestering glycogen masses; (e) are generally found to contain reaction product in preparations incubated from demonstration of acid phosphatase activity. The findings emphasize the role of the lysosomal system in the physiological process of postnatal glycogen mobilization and appear relevant in the interpretation of type II glycogen storage disease.     

狗在运动中的肌糖原消耗与无氧阈的关系

本实验测定了5条狗的无氧阈值,运动耐受时间、衰竭时的血乳酸浓度及运动中的肌糖原消耗量。结果如下:无氧阈值,1.与运动耐受时间呈正相关(r=0.947,P<0.02);2.与运动中肌糖原消耗量呈负相关(r=-0.959,P<0.01);3.与衰竭时的血乳酸浓度呈负相关(r=-0.942,P<0.02)。实验结果提示,无氧阈值是反映机体耐力的可靠指标。而运动中肌糖原消耗少,血乳酸积累程度轻,可能是无氧阈值之所以能够反映机体耐力的物质基础。     

ELECTRON MICROSCOPE RADIOAUTOGRAPHIC STUDY OF GLYCOGEN SYNTHESIS IN THE RABBIT RETINA

Glycogen is present in the rabbit retina in monoparticulate form. Beta particles (~ 229 A) are abundant in Müller cell cytoplasm, particularly in its inner portion, decreasing in number outwards along the cell. They are slightly larger (~ 250 A) and much scarcer in neurons, though regularly present in the juxtanuclear Golgi region of ganglion cells. When the retina was incubated in a glucose-free medium, it was rapidly depleted of native glycogen. On further incubation in medium containing glucose-³H plus unlabeled glucose, glycogen reappeared in the form of beta particles of the same size and distribution as native ones, while radioautography revealed the appearance of amylase-labile radioactivity in the same locations. This newly formed glycogen was not associated with any particular organelle. The rate of synthesis, as judged from the amount of radioactivity, was high in the inner portion of Müller cells and declined uniformly toward the cell outer end, following a logarithmic gradient. The rate of synthesis was low in ganglion cells, at best approaching values in the outer portion of Müller cells. The concentration of glycogen in the inner portion of Müller cells is consistent with the view that it may be the source of glucose for the anaerobic glycolysis prevailing in the inner retina.     

CHANGE IN THE GLYCOGEN CONTENT OF SEA URCHIN EGGS DURING EARLY DEVELOPMENT

During development of eggs of the sea urchins, Pseudocenrotus depressus and Anthocidaris crassispina , the glycogen level is maintained from the time of fertilization to the swimming blastula stage and then decreases rapidly in the early gastrula stage. During development of eggs of Clypeaster japonicus. Hemicentrotus pulcherrimus and Mespilia globulus the glycogen content decreases slowly from the time of fertilization to the mesenchyme blastula stage, and then more rapidly during gastrulation. The amounts of glycogen mobilized in the embryos from the time of fertilization to the morula stage correspond to 67% of the amount of O₂ consumed in Mespilia eggs, 62% in Clypeaster eggs, 30% in Hemicentrotus eggs and 0–4% in Anthocidaris and Pseudocentrotus eggs. The main energy source in early development seems to differ in different species. When eggs and embryos were incubated with [¹⁴C]glucose for 10min, considarable ¹⁴C-radioactivity accumulated in the glycogen fraction. The rate of [¹⁴C]glucose incorporation into glycogen increased gradually during the first 6 hr after fertilization (up to the morula stage), decreases during the next 4 hr (up to the early blastula stage), and then increased again.     

FACTORS AFFECTING THE TURNOVER OF CEREBRAL GLYCOGEN AND LIMIT DEXTRIN IN VIVO

The turnover of cerebral glycogen in mice has been investigated by using [U-¹⁴C]glucose as a precursor. The time required for turnover of total glycogen and limit dextrin has been determined in normal animals and animals given phenobarbital or hydrocortisone. In all 3 groups, the turnover time for limit dextrin was twice that of total glycogen. Phenobarbital increased the time for turnover of total glycogen and limit dextrin approximately 2-fold, whereas hydrocortisone diminished the turnover time of both fractions to one-half. The accumulation of glycogen during phenobarbital anesthesia (2·5-fold) is attributed to the decrease in rate of phosphorolysis rather than elevated glycogenesis. The ratio of phosphorylase a to total phosphorylase was significantly decreased in the brains of phenobarbital-treated mice, while the ratio of glycogen synthetase I to total synthetase activity was not affected. The administration of hydrocortisone had no effect on either the phosphorylase or synthetase of mouse brain. A mathematical model was devised to determine the rate constants for incorporation of labelled glucose into brain glycogen and the subsequent loss of radioactivity. Metabolite levels and enzyme activities have been correlated with the observed changes in glycogen turnover.     

孕期小鼠服用阿斯匹林后胎盘糖原组织化学的图像分析

为探讨孕期小鼠服用阿斯匹林后 ,胎盘糖原含量变化与胎鼠发育异常之间的关系 ,给妊娠 8.5天的小鼠连续 3天灌服阿斯匹林 (2 5 0 mg/ kg/ d) ,用组织化学方法、图像分析技术定量研究第 12 .5天和第 17.5天胎盘糖原的变化。结果显示 ,第 12 .5和 17.5天用药组海绵滋养层细胞特别是糖原细胞内糖原含量与对照组相比显著减少 (P<0 .0 0 1,P<0 .0 0 1) ,胚胎发育异常的胎盘其糖原细胞的形态结构及糖原含量均有明显改变。提示阿斯匹林可通过降低胎盘的糖原贮存 ,使其葡萄糖释放量下降 ,进而使胚胎发育过程中所必需的能量供应不足 ,影响了胎鼠的正常发育甚至导致畸形发生     

ISOLATION, ULTRASTRUCTURE, AND BIOCHEMICAL CHARACTERIZATION OF GLYCOGEN IN INSECT FLIGHT MUSCLE

Glycogen from flight muscle of the blowfly, Phormia regina, has been characterized ultrastructurally and biochemically. In situ, glycogen is in the form of rosettes, which vary in size with diameters of up to 0.1 µ. Sedimentation analysis of pure glycogen, isolated by mild buffer extraction, reveals a polydisperse molecular weight spectrum, with larger particles having molecular weights of 100 million. Treatment of native glycogen with alkali, under conditions usual for the extraction of the polysaccharide from tissues, results in a 5- to 10-fold reduction in molecular weight, as well as a chemical alteration of the molecule. Flight muscle phosphorylase has a lower affinity for native than for alkali-treated glycogen. The maximum velocity of the enzyme is also lower with native substrate. The apparent K_m for inorganic phosphate is higher with native glycogen as cosubstrate. These kinetic differences with native and partially degraded glycogen demonstrate the importance of using the natural substrate in studies of biochemical control mechanisms.     

THE LOCALIZATION OF GLYCOGEN IN THE SPERMATOZOA OF VARIOUS INVERTEBRATE AND VERTEBRATE SPECIES

With the periodic acid-thiosemicarbazide-silver proteinate procedure for the detection of polysaccharides in thin sections, glycogen is localized in the cavities of centrioles and basal bodies, within the axoneme (and surrounding it), in mitochondria, and in the "packing" cytoplasm of the middle piece of spermatozoa of several invertebrate and vertebrate species. The cytochemical localization of glycogen is verified by extraction with α-amylase solution. These findings establish the existence of stored glycogen in sperm. The polysaccharide presumably serves as an endogenous source of energy in the absence of extracellular metabolites, under either aerobic or anaerobic conditions. Other hypotheses on the physiological significance of intracellular glycogen stores in sperm are discussed. Sperm that store glycogen contain some enzymes of glycogen metabolism. In the presence of glucose-1-phosphate, ATP, and Mg⁺⁺ ions, an amylophosphorylase catalyzes the in vivo synthesis of glycogen. The newly formed product resembles γ-particles, and is digestible with α-amylase.     

BRAIN GLYCOGEN AFTER INTRACISTERNAL INSULIN INJECTION

Abstract— Intracisternal injection of 0·1 i.u. of insulin to rats caused an increase in the brain glycogen content. Intravenous and intraperitoneal injection of the same amount had no effect on brain glycogen. The increase after intracisternal injection was first observed after 3–4 hr.     

ELECTRON MICROSCOPIC STUDIES ON THE BEHAVIOR OF GLYCOGEN PARTICLES DURING OOGENESIS IN THE SEA URCHIN

The behavior of glycogen particles during oogenesis in the sea urchin was studied by electron microscopy. Before the beginning of oogenesis the nurse cells include many glycogen particles, which are spherical or multiangular in shape and about 600 A in diameter, lying within the vesicle of the large granules and also in the cytoplasm among the granules. There are few glycogen particles in the spaces among the oocytes and the nurse cells. At the early stage of oogenesis the limiting membrane of the large granule breaks locally and the glycogen particles in the vesicle are dispersed into the cytoplasm. The plasma membrane of the nurse cell also breaks in places and glycogen particles are spread throughout the intercellular space. At the beginning of vitellogenesis, β-pinosomes begin to be formed at the periphery of the oocyte; these take in glycogen particles from the outside which are progressively broken into smaller units.