Intranuclear synthesized and native glycogen particles in human gastric cancer: Ultrastructure and histochemistry

Summary Ultrastructural and histochemical studies on human gastric cancer cells disclosed the presence of native and synthesized glycogen particles. The glycogen particles were investigated in the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system and non-incubated native glycogen in human gastric adenocarcinoma tubulare.It was observed that focal synthesis localized in the intracytoplasmic matrix and intranucleus. Intranuclear synthesized glycogen appeared as a rosette form ranging from 1100 to 1300 Å in diameter and free particles ranging from 325 to 900 Å in diameter. The synthesis of glycogen appeared in the nucleus as well as in the cytoplasm of the human gastric cancer cells, and the synthesized glycogen was observed as a group of particles. Newly formed glycogen particles appeared occasionally in the interchromatin area as a large macromolecular structure of rosette form.Native glycogen appeared as a free-particle (250–333 Å, medium=300 Å) and aggregated rosette from (694–1050 Å, medium=917 Å) in the autophagosome of gastric cancer cells. There was not, however, a native glycogen particle in the nuclei of gastric cancer cells.Under certain conditions the nuclei of gastric cancer cells can acquire the capacity to synthesize glycogen.     

Polyglucose particles histochemically synthesized by glycogen synthetase activity in the living chick retina

Summary Electron histochemical techniques for glycogen synthetase has been applied to the living retina of the chick and the polyglucose particles synthesized from UDPG in the paraboloid of the accessory cone were compared with those synthesized by the conventional histochemical techniques.In the retinal incubated in the medium for glycogen synthetase in vivo, synthesized polyglucose particles were located in the cytoplasmic matrices and most of the particles were less than 200 Å in diameter. These particles were rather well stainable with lead citrate and filled the cytoplasmic matrices. However, the tubular structures were not flattened, but slightly dilated. Compared with polyglucose particles synthesized in vitro by glycogen synthetase, those demonstrated by the in vivo histochemical techniques showed closer resemblance to native glycogen particles in size and stainability with lead citrate.The polyglucose particles synthesized from UDPG by glycogen synthetase were apparently different from those synthesized from glucose-1-phosphate by phosphorylase and branching glycosyltransferase.     

Increased glycogen storage in yeast results in less branched glycogen

Glycogen is a branched polymer of glucose, synthesized as a reserve of both energy and carbon. The branched nature of glycogen is important for its function and polyglucosan bodies, particles that contain a glycogen-like polymer with reduced branching, are a feature of several disease states. The degree of glycogen branching is thought to be governed by the balance between glycogen synthesis and branching activities. However, there have been reports that the intrinsic properties of individual branching enzymes govern the degree of branching. To address the relationship between synthesis and branching more fully, we made use of the yeast Saccharomyces cerevisiae. The glycogen content of yeast cells was manipulated by using different growth conditions or by the introduction of specific mutations. Whenever glycogen storage was elevated, the polysaccharide formed was found to be less branched but normal branching could be restored by overexpression of branching enzyme.     

Ordered synthesis and mobilization of glycogen in the perfused heart

The molecular order of synthesis and mobilization of glycogen in the perfused heart was studied by 13C NMR. By varying the glucose isotopomer ([1-13C]glucose or [2-13C]glucose) supplied to the heart, glycogen synthesized at different times during the perfusion was labeled at different carbon sites. Subsequently, the in situ mobilization of glycogen during ischemia was observed by detection of labeled lactate derived from glycolysis of the glucosyl monomers. When [1-13C]glucose was given initially in the perfusion and [2-13C]glucose was given second, [2-13C]lactate was detected first during ischemia and [3-13C]lactate second. This result, and the equivalent result when the glucose labels were given in the reverse order, demonstrates that glycogen synthesis and mobilization are ordered in the heart, where glycogen is found morphologically only as beta particles. Previous studies of glycogen synthesis and mobilization in liver and adipocytes [Devos, P., & Hers, H.-G. (1979) Eur. J. Biochem. 99, 161-167; Devos, P., & Hers, H.-G. (1980) Biochem. Biophys. Res. Commun. 95, 1031-1036] have suggested that the organization of beta particles into alpha particles was partially responsible for ordered synthesis and mobilization. The observations reported here for cardiac glycogen suggest that another mechanism is responsible. In addition to examining the ordered synthesis and mobilization of cardiac glycogen, we have selectively monitored the NMR properties of 13C-labeled glycogen synthesized early in the perfusion during further glycogen synthesis from a second, differently labeled substrate. During synthesis from the second labeled glucose monomer, the glycogen resonance from the first label decreased in integrated intensity and increased in line width.(ABSTRACT TRUNCATED AT 250 WORDS)     

Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.     

An ultrastructural study of the craniocaudal continuation of the glycogen body

An ultrastructural analysis of the chicken glycogen body and its craniocaudal continuation areas shows a continuum of astroglial cell types. Characteristic glycogen body astroglia are confined to the classically defined body located in the chicken lumbosacral spinal cord. These are large cells which have an eccentric dark nucleus surrounded by a rim of dense cytoplasm which contains the usual complement of organelles. The remainder of the cell volume is occupied by alpha and beta glycogen particles interspersed with a flocculo-granular material continuous with the main cytoplasmic mass. Astroglial cells of continuation areas usually have a light cytoplasm and a centrally placed nucleus. They contain beta glycogen particles of varying sizes, but like the glycogen body cells, may have beta particles as large as 45 nm. Such particles, which resemble four leaf clovers in shape, are suggestive of an ordered substructure. Gliofilaments are not always conspicuous in astroglial perikarya, but large numbers of them are present in the processes. Although the continuation areas are mostly confined to gray matter regions, the contained astroglial processes exhibit circular, triangular, or cylindrical shapes and form an unpatterned mosaic. Astrocytic processes forming the glia limitans on the anterior and posterior margins of the cord often contain conspicuous amounts of glycogen. The ultrastructural identification of such large amounts of glycogen within the chicken nervous system suggests that it plays a major role in avian neural metabolism.     

Ultrastructural localization of glycogen in erythrocytes and developing erythrocytic cells in normal human bone marrow

Summary The periodic acid-thiosemicarbazide-silver proteinate (PA-TSC-SP) reaction was employed for the ultrastructural cytochemical localization of saliva-labile glycogen in the erythrocytic cells in normal human blood and bone marrow. Particulate glycogen was demonstrated in the cytoplasm of all developmental forms of erythrocytic cells from the proerythroblast through the reticulocyte; a few particles of glycogen also were present in mature erythrocytes even in the peripheral blood. Statistical evaluation of the number of glycogen particles in mid-plane cell sections at each morphological stage of development indicated a significant and stepwise decrease during cellular maturation. This change in glycogen content may reflect both cellular utilization and mitosis during the maturational sequence.Supported by Grant No. SR01AM 12084-15 from the National Institutes of Health, Bethesda, Maryland.Appreciation is expressed to Anita Topson, Barbara Speakmon and Marjorie Griffith for their technical assistance and to Dr. Gerald King for performing the bone marrow aspirations.     

SERGE, the subcellular site of initial hepatic glycogen deposition in the rat: a radioautographic and cytochemical study

Hormonal control of hepatic glycogen and blood glucose levels is one of the major homeostatic mechanisms in mammals: glycogen is synthesized when portal glucose concentration is sufficiently elevated and degraded when glucose levels are low. We have studied initial events of hepatic glycogen synthesis by injecting the synthetic glucocorticoid dexamethasone (DEX) into adrenalectomized rats fasted overnight. Hepatic glycogen levels are very low in adrenalectomized rats, and DEX causes rapid deposition of the complex carbohydrate. Investigation of the process of glycogen deposition was performed by light and electron microscopic (EM) radioautography using [3H]galactose as a glycogen precursor. Rats injected with DEX for 2-3 h and [3H]galactose one hour before being killed displayed an increasing number of intensely labeled hepatocytes. EM radioautography revealed silver grains over small (+/- 1 micron) ovoid or round areas of the cytosome that were rich in smooth endoplasmic reticulum (SER) and contained a high concentration of small dense particles. These distinct areas or foci of SER and presumptive glycogen (SERGE) were most numerous during initial periods of glycogen synthesis. After longer exposure to DEX (4-5 h) more typical deposits of cytoplasmic glycogen were evident in the SERGE regions. Several criteria indicated that the SERGE foci contained glycogen or presumptive glycogen: resemblance of the largest dense particles to beta-glycogen particles in EM; association of 3H-carbohydrate with the foci; removal of particles and label with alpha-amylase; and positive reaction with periodic acid-chromic acid-silver methenamine. The concentration of SER in the small foci and the association of newly formed glycogen particles with elements of SER suggest a role for this organelle in the initial synthesis of glycogen.     

Comparative study of nuclear and cytoplasmic glycogen isolated from mutant HD33 ascites cells

Mutant cells of the HD33 subline of the Ehrlich-Lettré ascites tumor synthesize and store glycogen mainly intranuclearly, when growing in vivo, and exclusively in the cytoplasm, when permanently cultivated as a suspension cell strain. To investigate whether there exist differences between glycogen of nuclear and cytoplasmic origin, the ultrastructure and the biophysical and biochemical properties of glycogen from in vivo and in vitro grown HD33 ascites cells were compared. Pronounced heterogeneity and differences in glycogen particle ultrastructure were evident in situ and after isolation of the native, high-molecular polysaccharide. Nuclear glycogen contains a fraction of heavier molecules (up to 2 X 10(9)) and larger particles (up to 340 nm) which could not be found in the cytoplasmic preparations, which contained only particles smaller than 140 nm. The subparticles of beta-type are similar in both nuclear and cytoplasmic glycogen. The absorption spectra and glucose analysis after degradation with phosphorylase and debranching enzyme indicate that nuclear glycogen has a higher degree of branching, associated with a decrease in the average chain length between the branching points, and shorter external polyglucosidic chains than cytoplasmic glycogen. This is the first report about the analysis and properties of isolated nuclear glycogen.     

Characteristic properties of the complex of glycogen synthase with glycogen

The glycogen synthase I--glycogen complex isolated from rabbit skeletal muscles is stable during precipitation with trichloroacetic acid and Sepharose 2B chromatography. The complex catalyzes the synthesis (lengthening) of the alpha-1.4-glucosyl chains when endogenous or exogenous enzyme-free glycogen is used, the initial rates of this synthesis being identical. Preincubation with glycogen does not cause activation of the complex or formation of additional glycogen synthase I--polysaccharide bonds. The complex is characterized by saturation with respect to glycogen; the molar concentration ratios of the non-reducible chain and protein monomer within the complex does not exceed 100. An increase in the length of the synthesized alpha-1.4-glycosyl chains of glycogen results in a decrease of the rate of the glycogen synthase reaction in time.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

厌氧氨氧化菌内含物——糖原颗粒

厌氧氨氧化菌(anaerobic ammonium-oxidizing bacteria, AnAOB)是分类学上新近建立的细菌类群。由于生长缓慢，培养困难，迄今没有获得纯培物。与已知细菌类群相比，AnAOB具有诸多特异性细胞结构和功能。AnAOB是化能自养型细菌，但在其细胞内经常可见贮藏性的内含物——糖原颗粒。探讨这种糖原颗粒的性状与动态，可深化人们对AnAOB的认识。本文结合文献报道及前期研究基础，对厌氧氨氧化菌糖原颗粒的结构、代谢和功能特性进行了探讨，分析认为AnAOB糖原颗粒分布于核糖细胞质内，且处于多途径合成与多位点消耗的动态平衡中；此外，糖原颗粒具有提供能量、碳架和应激保护等能力，对逆境下AnAOB的生存具有重要意义。本综述可为厌氧氨氧化菌的深入研究和工程应用提供支撑。     

Malin decreases glycogen accumulation by promoting the degradation of protein targeting to glycogen (PTG)

Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin. A hallmark of LD is the accumulation of insoluble glycogen in the cytoplasm of cells from most tissues. Glycogen metabolism is regulated by phosphorylation of key metabolic enzymes. One regulator of this phosphorylation is protein targeting to glycogen (PTG/R5), a scaffold protein that binds both glycogen and many of the enzymes involved in glycogen synthesis, including protein phosphatase 1 (PP1), glycogen synthase, phosphorylase, and laforin. Overexpression of PTG markedly increases glycogen accumulation, and decreased PTG expression decreases glycogen stores. To investigate if malin and laforin play a role in glycogen metabolism, we overexpressed PTG, malin, and laforin in tissue culture cells. We found that expression of malin or laforin decreased PTG-stimulated glycogen accumulation by 25%, and co-expression of malin and laforin abolished PTG-stimulated glycogen accumulation. Consistent with this result, we found that malin ubiquitinates PTG in a laforin-dependent manner, both in vivo and in vitro, and targets PTG for proteasome-dependent degradation. These results suggest an additional mechanism, involving laforin and malin, in regulating glycogen metabolism.     

Interrelationship of glycogen metabolism and lactose synthesis in mammary epithelial cells of mice.

Glycogen metabolism in mammary epithelial cells was investigated (i) by studying the conversion of glucose into glycogen and other cellular products in these cells from virgin, pregnant and lactating mice and (ii) by assaying the enzymes directly involved with glycogen metabolism. We find that: (1) mammary epithelial cells synthesized glycogen at rates up to over 60% that of the whole gland; (2) the rate of this synthesis was modulated greatly during the reproductive cycle, reaching a peak in late pregnancy and decreasing rapidly at parturition, when abundant synthesis of lactose was initiated; (3) glycogen synthase and phosphorylase activities reflected this modulation in glycogen metabolism; (4) lactose synthesis reached a plateau during late pregnancy, even though lactose synthase is reported to increase in the mouse mammary gland at this time. We propose that glycogen synthesis restricts lactose synthesis during late pregnancy by competing successfully for the shared UDP-glucose pool. The physiological advantage of glycogen accumulation during late pregnancy is discussed.     

Multiple requirements for glycogen synthesis by hepatocytes isolated from fasted rats

Glycogen synthesis from various combinations of substrates by hepatocytes isolated from rats fasted 24 h was studied. As reported by Katz et al. (Katz, J., Golden, S., and Wals, P. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3433-3437), appreciable rates of glycogen synthesis occurred only in the presence of gluconeogenic precursors and one of several amino acids, which includes L-glutamine. L-Leucine had negligible effects on glycogen synthesis from 20 mM dihydroxyacetone and/or 15 mM glucose when L-glutamine was not added to the medium. In the presence of 10 mM L-glutamine, L-leucine greatly increased glycogen synthesis from these substrates. alpha-Ketoisocaproate was ineffective, as was oleate. NH4Cl depressed glycogen synthesis from 10 mM glucose plus 20 mM dihydroxyacetone in the absence of added L-glutamine and enhanced that in its presence, but these effects were weak compared to those of L-leucine. The amino acid analogues L-norvaline and L-norleucine exerted effects that were similar to those exerted by L-leucine. Under all conditions studied, cycloheximide and puromycin inhibited net glycogen synthesis. Cycloheximide did not stimulate gluconeogenesis from dihydroxyacetone, or phosphorylase in hepatocytes from starved rats, or glycogenolysis in hepatocytes from fed rats. Puromycin, however, stimulated glycogenolysis in hepatocytes from fed rats. Glycogen synthesis from 20 mM dihydroxyacetone proceeds with a pronounced initial lag phase that can be shortened by incubation of cells with glutamine plus leucine before addition of dihydroxyacetone. Concurrent measurements of glycogen synthesis, glycogen synthase, and gluconeogenesis under different conditions reveal that in addition to protein synthesis, activation of glycogen synthase, which must occur to allow glycogen synthesis in hepatocytes, requires a second component which can be satisfied by addition of dihydroxyacetone or fructose to the cells.     

Molecular structural differences between type-2-diabetic and healthy glycogen

Glycogen is a highly branched glucose polymer functioning as a glucose buffer in animals. Multiple-detector size exclusion chromatography and fluorophore-assisted carbohydrate electrophoresis were used to examine the structure of undegraded native liver glycogen (both whole and enzymatically debranched) as a function of molecular size, isolated from the livers of healthy and db/db mice (the latter a type 2 diabetic model). Both the fully branched and debranched levels of glycogen structure showed fundamental differences between glycogen from healthy and db/db mice. Healthy glycogen had a greater population of large particles, with more α particles (tightly linked assemblages of smaller β particles) than glycogen from db/db mice. These structural differences suggest a new understanding of type 2 diabetes.     

Pathways of glycogen synthesis in Novikoff ascites-hepatoma cells

Affinity of glucose, fructose and mannose for tumour hexokinase and their rates of phosphorylation at saturation concentration have been correlated with rates of glycogen synthesis by intact tumour cells at different concentrations of the three substrates. Competition experiments with one sugar labelled and the other sugar unlabelled indicate inhibition of glycogen synthesis by the sugar with a low K(m) for hexokinase. Glycogen synthesis from glucose 1-phosphate in aged cells and from nucleoside in freshly prepared cells is stimulated by fructose and inhibited by glucose. The decrease in glycogen formation from glucose 1-phosphate by oligomycin is partially overcome by increased fructose concentrations. These results are explained by an activation of alpha-glucan phosphorylase by fructose and an inhibition of this enzyme by glucose. It is suggested that differences in localization of glucose 6-phosphate, available to the intact cell in various ways, determine its transformation into glycogen by either the UDP-glucose-alpha-glucan glucosyltransferase reaction or by the alpha-glucan phosphorylase reaction.     

Role of nuclear glycogen synthase and cytoplasmic UDP glucose pyrophosphorylase in the biosynthesis of nuclear glycogen in HD33 Ehrlich-Lettre ascites tumor cells

Biochemical and autoradiographic evidence show both glycogen synthesis and the presence of glycogen synthase (UDP glucose [UDPG]: glycogen 4-alpha-D-glucosyltransferase; EC 2.4.1.11) in isolated nuclei of Ehrlich-Lettré mouse ascites tumor cells of the mutant subline HD33. 5 d after tumor transplantation, glycogen (average 5-7 pg/cell) is stored mainly in the cell nuclei. The activity of glycogen synthase in isolated nuclei is 14.5 mU/mg protein. At least half of the total cellular glycogen synthase activity is present in the nuclei. The nuclear glycogen synthase activity exists almost exclusively in its b form. The Km value for (a + b) glycogen synthase is 1 x 10(-3) M UDPG, the activation constant is 5 x 10(-3) M glucose-6-phosphate (Glc-6-P). Light and electron microscopic autoradiographs of isolated nuclei incubated with UDP-[1-3H]glucose show the highest activity of glycogen synthesis not only in the periphery of glycogen deposits but also in interchromatin regions unrelated to detectable glycogen particles. Together with earlier findings on nuclear glycogen synthesis in intact HD33 ascites tumor cells (Zimmermann, H.-P., V. Granzow, and C. Granzow. 1976. J. Ultrastruct. Res. 54:115-123), the results of tests on isolated nuclei suggest a predominantly appositional mode of nuclear glycogen deposition, without participation of the nuclear membrane system. In intact cells, synthesis of UDPG for nuclear glycogen synthesis depends on the activity of the exclusively cytoplasmic UDPG pyrophosphorylase (UTP: alpha-D-glucose-1-phosphate uridylyltransferase; EC 2.7.7.9). However, we conclude that glycogen synthesis is not exclusively a cytoplasmic function and that the mammalian cell nucleus is capable of synthesizing glycogen.     

Partly ordered synthesis and degradation of glycogen in cultured rat myotubes.

The following questions concerning glycogen synthesis and degradation were examined in cultured rat myotubes. 1) Is synthesis and degradation of the individual glycogen molecule a strictly ordered process, with the last glucosyl unit incorporated into the molecule being the first to be released (the last-in-first-out principle), or is it a random process? 2) Are all glycogen molecules in skeletal muscle synthesized and degraded in phase (simultaneous order) or out of phase (sequential order)? Basal glycogen stores were minimized by fasting and were subsequently replenished in two intervals, the first (0-0.5 h) with tritium-labeled and the second (0.5-3 h) with carbon-labeled glucose as precursor. Glycogen degradation was initiated by addition of forskolin. The kinetics of glycogen accumulation as well as degradation could be approximated by monoexponential equations with rate constants of 0.81 and 1.39 h(-1), respectively. The degradation of glycogen largely followed the last-in-first-out principle, particularly in the initial period. Analysis of the size of the glycogen molecules and the beta-dextrin limit during glycogen accumulation and degradation showed that both synthesis and degradation of glycogen molecules are largely sequential and the small deviation from this order is most pronounced at the beginning of the accumulation and at the end of the degradation period. This pattern may reflect the number of synthase and phosphorylase molecules and fits well with the role of glycogen in skeletal muscle as a readily available energy store and with the known structure of the glycogen molecule. It is emphasized that the observed nonlinear relation between the change in glycogen concentration and release of label during glycogen degradation may have important practical consequences for interpretation of experimental data.