Glycogen Particles Research Articles

Muscle Biopsy as a Diagnostic Tool in Pompe Disease

Today, myopathological, biochemical and genetic characteristics of Pompe disease are well established, and many reports have been published following the first very precisely documented myopatholgical description of adultonset Pompe disease in 1968. The clinical awareness and need for diagnostic precision of Pompe disease has increased since enzyme replacement therapy (ERT) has become widely available. Primary characteristic myopathological findings of Pompe disease are rounded vacuoles that are irregular in shape and size. All infantile biopsies show pronounced vacuolisation of almost all fibres. In juvenile biopsies, the percentage of vacuolated fibres decreases to 75%, and in most adult patients only 10–50% of the fibres reveal vacuolisation. In infantile cases, both fibre types are equally affected, while in juvenile and adult cases a predominant vacuolisation of type 2 fibres occurs. The second myopathological hallmark is periodic acid-Schiff (PAS)– positive material packed in these vacuoles, sometimes lost due to fixation procedures and therefore sometimes only notable as optically empty vacuolar spaces. Thirdly, acid phosphates histochemistry reveals positive staining in vacuolated fibres, indicating lysosomal dysfunction. In addition to these classic findings, autophagic vacuoles, sometimes containing heterogeneous material including cytoplasmic degradation products and myeloid structures, are seen at all ages, especially in type 2 fibres. Ultra-structurally, these autophagic vacuoles contained particulate glycogen and/or autophagic debris, including myelin-like figures. Almost all samples show lipofuscin accumulation in the area of lysosomal residual bodies and rarely lipid droplets as a sign of lysosomal ageing. Infrequently, single rimmed vacuoles are notable. Fibre splitting is an uncommon aspect. Due to excessive fibre vacuolisation, a subsarcolemmal packing of mitochondria is notable in most cases. Single fibre necrosis, basophilia and phagocytosis may be present in biopsies of all ages. Furthermore, mild fibrosis and fatty degeneration may be found in about 10% of the biopsies. Immunohistochemistry does not normally present signs of an inflammatory myopathy. In selected biopsies, some macrophages may be noticed, and vacuoles may contain caveolin-3 and dysferlin positive membrane debris. Although all classic hallmarks of Pompe disease are well documented over several decades, Pompe disease in adult-onset patients may still be missed histologically, especially when a clinically unaffected muscle is chosen for biopsy. Consequently, in modern diagnostics, dry blood spot (DBS) testing followed by -glucosidase (GAA) gene nalysis helps to overcome this muscle biopsy neglect. However, in some adult patients presenting with a blurred imb girdle weakness and atrophy, combined muscle MRI, light and electron microscopy, and biochemical analysis ay be still essential for making an accurate diagnosis of Pompe disease.

Processivity and Subcellular Localization of Glycogen Synthase Depend on a Non-catalytic High Affinity Glycogen-binding Site

Glycogen synthase, a central enzyme in glucose metabolism, catalyzes the successive addition of α-1,4-linked glucose residues to the non-reducing end of a growing glycogen molecule. A non-catalytic glycogen-binding site, identified by x-ray crystallography on the surface of the glycogen synthase from the archaeon Pyrococcus abyssi, has been found to be functionally conserved in the eukaryotic enzymes. The disruption of this binding site in both the archaeal and the human muscle glycogen synthases has a large impact when glycogen is the acceptor substrate. Instead, the catalytic efficiency remains essentially unchanged when small oligosaccharides are used as substrates. Mutants of the human muscle enzyme with reduced affinity for glycogen also show an altered intracellular distribution and a marked decrease in their capacity to drive glycogen accumulation in vivo. The presence of a high affinity glycogen-binding site away from the active center explains not only the long-recognized strong binding of glycogen synthase to glycogen but also the processivity and the intracellular localization of the enzyme. These observations demonstrate that the glycogen-binding site is a critical regulatory element responsible for the in vivo catalytic efficiency of GS.

Intracellular compartmentalization of skeletal muscle glycogen metabolism and insulin signalling

The interest in skeletal muscle metabolism and insulin signalling has increased exponentially in recent years as a consequence of their role in the development of type 2 diabetes mellitus. Despite this, the exact mechanisms involved in the regulation of skeletal muscle glycogen metabolism and insulin signalling transduction remain elusive. We believe that one of the reasons is that the role of intracellular compartmentalization as a regulator of metabolic pathways and signalling transduction has been rather ignored. This paper briefly reviews the literature to discuss the role of intracellular compartmentalization in the regulation of skeletal muscle glycogen metabolism and insulin signalling. As a result, a hypothetical regulatory mechanism is proposed by which cells could direct glycogen resynthesis towards different pools of glycogen particles depending on the metabolic needs. Furthermore, we discuss the role of skeletal muscle transverse tubules as potential modulators of tissue insulin responsiveness.

Differential pattern of glycogen accumulation after protein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression, compared to PPP1R3C and PPP1R3A, in skeletal muscle cells

BackgroundPPP1R6 is a protein phosphatase 1 glycogen-targeting subunit (PP1-GTS) abundant in skeletal muscle with an undefined metabolic control role. Here PPP1R6 effects on myotube glycogen metabolism, particle size and subcellular distribution are examined and compared with PPP1R3C/PTG and PPP1R3A/GM.ResultsPPP1R6 overexpression activates glycogen synthase (GS), reduces its phosphorylation at Ser-641/0 and increases the extracted and cytochemically-stained glycogen content, less than PTG but more than GM. PPP1R6 does not change glycogen phosphorylase activity. All tested PP1-GTS-cells have more glycogen particles than controls as found by electron microscopy of myotube sections. Glycogen particle size is distributed for all cell-types in a continuous range, but PPP1R6 forms smaller particles (mean diameter 14.4 nm) than PTG (36.9 nm) and GM (28.3 nm) or those in control cells (29.2 nm). Both PPP1R6- and GM-derived glycogen particles are in cytosol associated with cellular structures; PTG-derived glycogen is found in membrane- and organelle-devoid cytosolic glycogen-rich areas; and glycogen particles are dispersed in the cytosol in control cells. A tagged PPP1R6 protein at the C-terminus with EGFP shows a diffuse cytosol pattern in glucose-replete and -depleted cells and a punctuate pattern surrounding the nucleus in glucose-depleted cells, which colocates with RFP tagged with the Golgi targeting domain of β-1,4-galactosyltransferase, according to a computational prediction for PPP1R6 Golgi location.ConclusionsPPP1R6 exerts a powerful glycogenic effect in cultured muscle cells, more than GM and less than PTG. PPP1R6 protein translocates from a Golgi to cytosolic location in response to glucose. The molecular size and subcellular location of myotube glycogen particles is determined by the PPP1R6, PTG and GM scaffolding.

Ultrastructural and cytochemical study on vitellogenesis in the diphyllidean cestode Echinobothrium euterpes (Echinobothriidae) and its phylogenetical implications

AbstractThe first description of vitellogenesis in the Diphyllidea is presented in this paper. Though the type of vitellogenesis and mature vitellocyte in Echinobothrium euterpes appear to be unique among the Eucestoda, however, they somewhat resemble that observed in the two orders of the lower cestodes, Tetraphyllidea and Proteocephalidea. Vitellocyte maturation is characterized by: (1) an increase in cell volume; (2) extensive development of short, parallel, frequently concentric cisternae of GER that produce dense proteinaceous granules; (3) development of Golgi complexes engaged in packaging this material; (4) progressive formation of saturated lipid droplets; their continuous enlargement and fusion; (5) formation of small accumulations of glycogen particles scattered between and among lipid droplets in the cytoplasm of maturing vitellocytes; (6) concentration of dense proteinaceous granules in the peripheral layer of cytoplasm, around the cell plasma membrane; and (7) vacuolization of cytoplasm of mature vitellocytes accompanied by a rapid increase in its volume. A new, unreported type of dense proteinaceous granules, situated around the limiting plasma membranes of mature vitellocytes, is described. Vitellogenesis evidently differs from that with typical shell-globules and shell-globule clusters previously reported in other taxa of lower cestodes. Cytochemical staining with periodic acidthiosemicarbazide-silver proteinate for glycogen indicates a strongly positive reaction for glycogen particles between and around large unsaturated lipid droplets of the maturing and mature vitellocytes. Some hypotheses concerning the interrelationship between this pattern of vitellogenesis, possible mode of egg formation, embryonic development and diphyllidean life cycle, and their phylogenetic implications are drawn and discussed.

Changes in Glycogen and Glycosaminoglycan Levels in Hepatocytes of Iduronate-2-Sulfatase Knockout Mice before and after Recombinant Iduronate-2-Sulfatase Supplementation

PurposeMucopolysaccharidosis II (MPS II) is a lysosomal storage disorder caused by a deficiency of iduronate-2 sulfatase (IdS), which is involved in the degradation of glycosaminoglycan (GAG). In this study, the frequency of fasting hypoglycemia in patients with MPS II was investigated and changes in accumulation of glycogen and GAG in the hepatocytes of IdS-knockout (KO) mice were evaluated before and after recombinant IdS enzyme replacement therapy (ERT).Materials and MethodsPlasma glucose levels were evaluated after an 8-hour fast in 50 patients with MPS II. The IdS-KO mice were divided into three groups (group 2; saline, group 3; 0.15 mg/kg of IdS, and group 4; 0.5 mg/kg of IdS); wild-type mice were included as controls (group 1). ERT was initiated intravenously at four weeks of age, and continued every week until 20 weeks of age.ResultsThe mean glucose level after an 8-hour fast was 94.1 ± 23.7 mg/dL in the patients with MPS II. Two (4%) out of 50 patients had fasting hypoglycemia. For the mice, GAG in the lysosomes nearly disappeared and glycogen particles in the cytoplasm were restored to the normal range in group 4.ConclusionGlucose metabolism in patients with MPS II appeared to function well despite hepatocytic GAG accumulation and hypothetical glycogen depletion. A higher dose of IdS infusion in MPS II mice led to disappearance of lysosomal GAG and restoration of glycogen to the cytoplasm of hepatocytes.

Three-Dimensional Architecture and Immuno-Histochemistry of Intermediate Filaments in Purkinje Cells in Sheep and Humans

It has been established that Purkinje cells in the bovine heart have abundant intermediate filaments positive for desmin antibody within their cytoplasm. However, there were no reports on three-dimensional architecture of intermediate filaments in Purkinje cells. In this study, Purkinje cells in sheep and human hearts were investigated by immuno-histochemistry, transmission and scanning electron microscopy (TEM and SEM). In TEM images, Purkinje cells in sheep hearts were considerably larger in size than working cardiac myocytes, and had a few peripherally-placed myofibrils and a great number of intermediate filaments in addition to glycogen particles. Intermediate filaments showed positive reactions for desmin antibody. Although Purkinje cells in humans were somewhat larger than working cardiac myocytes and had relatively abundant myofibrils, they also showed positive reactions. The saponin or NaOH treatment of cardiac tissues resulted in digestion of both glycogen particles and cytoplasmic matrix, and enabled us to view intermediate filaments by SEM. Intermediate filaments, which formed delicate, complicated networks, surrounded nuclei, mitochondria and myofibrils. In general, intermediate filaments in sheep Purkinje cells were distributed throughout the cytoplasm, while those in human Purkinje cells were concentrated mainly near the nucleus. In Purkinje fibers, it was sugested that a delicate network of intermediate filaments acted as the cytoskeleton to maintain stable propagation of the impulse.

Conditional ablation of glycogen synthase kinase 3β in postnatal mouse kidney

Glycogen synthase kinase (GSK)3 is a ubiquitously expressed serine/threonine kinase existing in two isoforms, namely GSK3α and GSK3β. Aside from the long-recognized role in insulin signal transduction and glycogen biosynthesis, GSK3β has been recently coined as a master control molecule in nuclear factor-κB activation and inflammatory kidney injury. Nevertheless, previous studies are less conclusive because they relied greatly on small molecule inhibitors, which lack selectivity and barely distinguish between the GSK3 isoforms. In addition, early embryonic lethality after global knockout of GSK3β precludes interrogation of the biological role of GSK3β in the adult kidney. To circumvent these issues, the Cre/loxP system was used to generate a conditional knockout mouse model in which the GSK3β gene was specifically deleted in kidney cortical tubules at postnatal mature stage. Kidney-specific ablation of GSK3β resulted in a phenotype no different from control littermates. Knockout mice (KO) were viable and exhibited normal development and normal kidney physiology in terms of kidney function, urine albumin excretion, and urine-concentrating ability. It is noteworthy that apart from normal glomerular and tubulointerstitial morphology, the kidneys from KO demonstrated more glycogen accumulation in the renal cortical tubules as assessed by both periodic acid-Schiff staining for light microscopy and direct biochemical assay, consistent with an elevated glycogen synthetic activity as evidenced by diminished inhibitory phosphorylation of glycogen synthase that occurred subsequent to GSK3β ablation. This finding was further validated by electron microscopic observations of increased deposition of glycogen particles in the renal tubules of KO, suggesting that GSK3α could not fully compensate for the loss of GSK3β in regulating glycogen metabolism in the kidney. Collectively, our study suggests that kidney-specific ablation of GSK3β barely affects kidney function and histology under normal circumstances. Extended examinations of these KO under diseased conditions are merited to understand the role of GSK3β in renal pathophysiology.

Ultrastructural study of naturally occuring ovine pulmonary adenocarcinoma in Fars province, Iran

Summary Ovine pulmonary adenocarcinoma (OPA) was studied in the lungs of 15 naturally-affected sheep (9 with classical and 6 atypical lesions) by transmission electron microscopy. Two lung samples from normal sheep were used to develop the ultrastructural criteria. Pathologic lesions consisted of focal-multifocal to coalescent nodules or masses in the cranioventral or diaphragmatic lobes. Ultrastructural characteristics of tumor cells in the alveoli and bronchioles allowed categorization of these cells into three groups; neoplastic alveolar type (AT) П cells in the alveoli contained numerous cytoplasmic lamellar bodies, well developed rough endoplasmic reticulum and glycogen particles; neoplastic clara cells in the bronchioles contained apical electron-dense granules and well-developed smooth endoplasmic reticulum; undifferentiated tumor cells in the alveoli and bronchioles lacked characteristic lamellar bodies or electron-dense granules. Neither complete virions nor viral inclusions were seen in the neoplastic cells. In the present study, there were no ultrastructural differences in the neoplastic cells between classical and atypical forms. It seems that type II pneumocytes and clara cells are the origin of the neoplasia in the alveoli and bronchioles, respectively.

Enhanced effects of nonisotopic hafnium chloride in methanol as a substitute for uranyl acetate in TEM contrast of ultrastructure of fungal and plant cells

This ultrastructural study showed that nonisotopic methanolic hafnium chloride and aqueous lead solution was an excellent new electron stain for enhancing TEM contrasts of fungal and plant cell structures. The ultrastructural definition provided by the new stain was often superior to that provided by conventional staining with uranyl acetate and lead. Definition of fine ultrastructure was also supported by quantitative data on TEM contrast ratios of organelles and components in fungal and plant cells. In particular, polysaccharides, which were localized in cell walls, glycogen particles, starch grains, and plant Golgi vesicle components, were much more reactive to the new stain than to the conventional one. The new nonisotopic stain is useful for enhancing the contrast of ultrastructure in biological tissues and is a safer alternative to uranyl acetate.

Energy sensing by the AMP-activated protein kinase and its effects on muscle metabolism

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status, and a regulator of energy balance at both the cellular and whole body levels. Although ubiquitously expressed, its function is best understood in skeletal muscle. AMPK contains sites that reversibly bind AMP or ATP, with an increase in cellular AMP:ATP ratio (signalling a fall in cellular energy status) switching on the kinase. In muscle, AMPK activation is therefore triggered by sustained contraction, and appears to be particularly important in the metabolic changes that occur in the transition from resistance to endurance exercise. Once activated, AMPK switches on catabolic processes that generate ATP, while switching off energy-requiring processes not essential in the short term. Thus, it acutely activates glucose uptake (by promoting translocation of the transporter GLUT4 to the membrane) and fatty acid oxidation, while switching off glycogen synthesis and protein synthesis (the later via inactivation of the mammalian target-of-rapamycin pathway). Prolonged AMPK activation also causes some of the chronic adaptations to endurance exercise, such as increased GLUT4 expression and mitochondrial biogenesis. AMPK contains a glycogen-binding domain that causes a sub-fraction to bind to the surface of the glycogen particle, and it can inhibit glycogen synthesis by phosphorylating glycogen synthase. We have shown that AMPK is inhibited by exposed non-reducing ends in glycogen. We are working on the hypothesis that this ensures that glycogen synthesis is rapidly activated when glycogen becomes depleted after exercise, but is switched off again as soon as glycogen stores are replenished.

Subcellular localization-dependent decrements in skeletal muscle glycogen and mitochondria content following short-term disuse in young and old men

Previous studies have shown that skeletal muscle glycogen and mitochondria are distributed in distinct subcellular localizations, but the role and regulation of these subcellular localizations are unclear. In the present study, we used transmission electron microscopy to investigate the effect of disuse and aging on human skeletal muscle glycogen and mitochondria content in subsarcolemmal (SS), intermyofibrillar (IMF), and intramyofibrillar (intra) localizations. Five young (∼23 yr) and five old (∼66 yr) recreationally active men had their quadriceps muscle immobilized for 2 wk by whole leg casting. Biopsies were obtained from m. vastus lateralis before and after the immobilization period. Immobilization induced a decrement of intra glycogen content by 54% (P < 0.001) in both age groups and in two ultrastructurally distinct fiber types, whereas the content of IMF and SS glycogen remained unchanged. A localization-dependent decrease (P = 0.03) in mitochondria content following immobilization was found in both age groups, where SS mitochondria decreased by 33% (P = 0.02), superficial IMF mitochondria decreased by 20% (P = 0.05), and central IMF mitochondria remained unchanged. In conclusion, our findings demonstrate a localization-dependent adaptation to immobilization in glycogen and mitochondria content of skeletal muscles of both young and old individuals. Specifically, this suggests that short-term disuse preferentially affects glycogen particles located inside the myofibrils and that mitochondria volume plasticity can be dependent on the distance to the fiber border.

Three-dimensional reconstruction of living mouse liver tissues using cryotechniques with confocal laser scanning microscopy

Soluble proteins and glycogen particles are well preserved in paraffin-embedded sections prepared by in vivo cryotechnique (IVCT) and cryobiopsy followed by freeze substitution fixation. We performed confocal laser scanning microscopic analyses on the distributions of glycogen with periodic acid-Schiff (PAS) staining and serum proteins with immunostaining for mouse liver tissues. Livers of fully fed mice showed a strong fluorescence signal of PAS staining in all hepatocytes and immunofluorescence of immunoglobulin kappa light chain (Igκ) in blood vessels and bile canaliculi. However, some hepatocytes in mechanically damaged livers were PAS-negative and Igκ-immunopositive, showing extraction of glycogen particles and infiltration of serum proteins in hepatocytes. By three-dimensional (3D) reconstruction of serial optical sections, interconnecting hepatic sinusoids and bile canaliculi were detected with Igκ immunostaining between trabecular hepatocytes that were PAS stained. In PAS-stained samples under fasting conditions, interstitial structures along sinusoids were clarified in vivo by 3D reconstruction because of the lower PAS staining intensity of hepatocytes. In addition, 100-μm-thick eosin-stained slices provided 3D structural images more than 30 μm in thickness away from tissue surfaces, showing blood vessels with flowing erythrocytes and networks of bile ducts and canaliculi. IVCT and cryobiopsy with histochemical analyses enabled us to visualize native hepatocytic glycogen and 3D structures, such as vascular networks, reflecting their living states by confocal laser scanning microscopy.

Effect of ivermectin on the integument and dorsoventral muscles of the tick Argas (Persicargas) persicus (Oken) (Ixodoidea: Argasidae)

Light and transmission electron microscopy (TEM) revealed that integument of Argas persicus consisted of cuticle underlined with epidermal cells. Cuticle consisted of outer epicuticle and inner procuticle. Epicuticle is further subdivided into thin wax, thin electron dense cuticulin, and thick less electron dense protein epicuticle layers. The procuticle consisted of exo-, endo-, and subcuticle. The procuticle contained numerous pore canals emerged from epidermal cells. Dermal glands were scattered between epidermal cells. TEM showed that each muscle cell contained two types of myofilaments and numerous electron dense bodies. Cytoplasmic organelles are peripherally located and plasma lemma invaginated deeply forming sarcotubular system. Feeding resulted in marked increase in cytoplasmic organelles and secretions of both epidermal cells and dermal glands. It also led to stretching of myofilaments, proliferation of cytoplasmic organelles, and appearance of glycogen particles in muscle cells. Subcutaneous inoculation of ivermectin (IVM) at a dose of 400 mug/kg pigeon resulted in extensive alterations in the integument and muscle cells. Both exhibited intense vacuolation of the cytoplasm, damage of cytoplasmic organelles, and swelling of the nucleus. It also caused aggregation of pore canals in the procuticle, depletion of secretory vesicles in dermal gland cells, and destruction of myofilaments, dense bodies, and sarcotubular system in muscle cells. The results suggest that IVM probably binds to the neurotransmitters or the hormones involved in secretion processes of epidermal cells and dermal glands in the integument or those involved in contraction of dorsoventral muscles.

Trophoblast glycogen cells differentiate early in the mouse ectoplacental cone: putative role during placentation

The role of differentiated trophoblast glycogen cells (GCs) in the ectoplacental cone (EPC) has not been elucidated yet. Recently, GC progenitors have been shown to be present from embryonic day 7.5 (E7.5), but glycogen is found in GC only from E10.5. Herein, we investigated the origin, localization and characterization of mouse GCs in EPC and their relationship with blood cells and trophoblast giant cells (TGCs) during placentation. Implantation sites (E5.5-E12.5) were processed for histological studies, histochemical detection (glycogen) and immunohistochemical staining (Ki67). Three-dimensional reconstruction of the EPC was obtained from suitably oriented embryos at E7.5. Our findings evidence that GCs are present and assembled in clusters from E6.5 to E12.5, and that they exhibit the classic vacuolated appearance and contain PAS-positive glycogen, which is amylase-sensitive and acetylation-resistant. In fact, only GCs were stained after acetylation, confirming unequivocally their presence in tissues. At E6.5, GCs showed numerous mitoses and vacuoles with scattered glycogen particles. At E7.5, GCs showed low numbers of mitoses and abundant vacuoles full of glycogen. During E7.5-E8.5, GCs were in close proximity to TGCs, and cells were intercalated by thin maternal blood spaces; placental GCs lost maternal blood contact during E9.5-E12.5. Our results indicate that GCs are originated and proliferate in the upper portion in the midregion of EPC at E6.5, and that at E7.5-E8.5 they show consistent glycogen deposits, which are likely metabolized to glucose. This compound may be directly transferred to circulating maternal blood, and used as a source of energy by GCs and TGCs during placentation.

Analysis of hepatic glycogen-associated proteins.

Glycogen particles are associated with a population of proteins that mediate its biological functions, including: management of glucose flux into and out of the glycogen particle, maintenance of glycogen structure and regulation of particle size, number, and cellular location. A survey of the glycogen-associated proteome would be predicted to identify the relative representation of known members of this population, and associations with unexpected proteins that have the potential to mediate other functions of the glycogen particle. We therefore purified glycogen particles from both mouse and rat liver, using different techniques, and analyzed the resulting tryptic peptides by MS. We also specifically eluted glycogen-binding proteins from the pellet using malto-oligosaccharides. Comparison of the rat and mouse populations, and analysis of specifically eluted proteins allow some conclusions to be made about the hepatic glycogen sub-proteome. With the exception of glycogen branching enzyme all glycogen metabolic proteins were detected. Novel associations were identified, including ferritin and starch-binding domain protein 1, a protein that contains both a transmembrane endoplasmic reticulum signal peptide and a carbohydrate-binding module. This study therefore provides insight into the organization of the glycogen proteome, identifies other associated proteins and provides a starting point to explore the dynamic nature and cellular distribution of this metabolically important protein population.

Hepatic differentiation of liver-derived progenitor cells and their characterization by microRNA analysis

We recently reported the isolation and characterization of a novel population of progenitor cells called liver-derived progenitor cells (LDPCs), which could differentiate into functional hepatocytes in vitro. However, our original studies resulted in relatively low and variable hepatic differentiation efficiency without validation of in vivo potential of LDPCs. Here, we report an efficient and robust hepatic differentiation of LDPCs under well-defined culture conditions and in vivo differentiation of LDPCs to mature hepatocytes. In addition to morphological studies, we performed reverse-transcription polymerase chain reaction (RT-PCR) and microRNA analyses of the in vitro hepatic differentiation of LDPCs to substantiate the efficiency of the differentiation process. The histological studies on the differentiated LDPCs showed that more than 50% of the cells were positive for albumin, cytokeratin 18, and hepatocyte nuclear factor 1 alpha and contained glycogen particles, all consistent with differentiation to functional hepatocytes. We also demonstrated by RT-PCR that upon differentiation, they expressed several markers found in mature hepatocytes and the microRNA profile of LDPCs became similar to the profile of fresh hepatocytes, confirming our morphological findings. Finally, the transplantation of LDPCs in a dipeptidyl peptidase IV-deficient (DPPIV(-/-)) rat model showed that LDPCs were able to engraft and form mature hepatocytes in the livers of the DPPIV(-/-) rats. In summary, LDPCs are a unique population of liver progenitor cells capable of hepatic differentiation both in vitro and in vivo, which makes them a potentially valuable resource for important applications such as pharmacological studies and cell therapies for a variety of liver disorders.

The subcellular localization of yeast glycogen synthase is dependent upon glycogen content.

The budding yeast, Saccharomyces cerevisiae, accumulates the storage polysaccharide glycogen in response to nutrient limitation. Glycogen synthase, the major form of which is encoded by the GSY2 gene, catalyzes the key regulated step in glycogen storage. Here, we utilized Gsy2p fusions to green fluorescent protein (GFP) to determine where glycogen synthase was located within cells. We demonstrated that the localization pattern of Gsy2-GFP depended upon the glycogen content of the cell. When glycogen was abundant, Gsy2-GFP was found uniformly throughout the cytoplasm, but under low glycogen conditions, Gsy2-GFP localized to discrete spots within cells. Gsy2p is known to bind to glycogen, and we propose that the subcellular distribution of Gsy2-GFP reflects the distribution of glycogen particles. In the absence of glycogen, Gsy2p translocates into the nucleus. We hypothesize that Gsy2p is normally retained in the cytoplasm through its interaction with glycogen particles. When glycogen levels are reduced, Gsy2p loses this anchor and can traffic into the nucleus.

Distribution and Development of Brown Adipocytes in the Murine and Human Adipose Organ

Murine white and brown adipocytes are found together in dissectible visceral and subcutaneous fat depots supplied by specific vessels and nerves, forming a multi-depot organ with plastic properties. Many of the anatomo-physiological features of murine fat depots apply to humans.

Histochemical approach of cryobiopsy for glycogen distribution in living mouse livers under fasting and local circulation loss conditions

Soluble proteins and glycogen particles, which are easily lost upon conventional chemical fixation, have been reported to be better preserved in paraffin-embedded sections by 'cryobiopsy' combined with freeze-substitution fixation (FS). In this study, we examined the distribution of glycogen in living mouse livers under physiologic and pathologic conditions with periodic acid-Schiff (PAS) staining by cryobiopsy. The livers of the fully fed mice showed high PAS-staining intensity in the cytoplasm of all hepatocytes. The PAS-staining intensity gradually decreased away from hepatocytes around portal tracts, depending on treatments with different alpha-amylase concentrations. At 6 or 12 h after fasting, PAS-staining intensity markedly decreased in restricted areas of zone I near the portal tracts. The cryobiopsy was repeatedly performed not only on different mice, but also on individuals. Next, glycogen distributions were evaluated by temporarily clipping of liver tissues of anesthetized mice, followed by recovery of blood circulation. In the liver tissues in which blood was recirculated for 1 h after the 30 min anoxia, PAS staining was still observed in zone II and also in restricted areas of zone I far from the portal tracts. In PAS-unstained hepatocytes, the immunoglobulin-kappa light chain was not detected in the cytoplasm, indicating that cell membrane permeability was retained and that glycogen metabolism was related to the functional state of blood circulation. We propose that the level of consumption or production of glycogen particles could vary in zone I, depending on the distance from the portal tracts. Thus, cryobiopsy combined with FS enabled us to examine time-dependent changes in glycogen distribution in the liver tissues of living mice. This combination might be applicable to the clinical evaluation of human liver tissues.