Massive Glycogen Accumulation Research Articles

GPR30 selective agonist G-1 induced insulin resistance in ovariectomized mice on high fat diet and its mechanism

BackgroundPrevious in vivo and in vitro studies have demonstrated that estrogen receptor agonist G-1 regulates glucose and lipid metabolism. This study focused on the effects of G-1 on cardiometabolic syndrome and anti-obesity under a high fat diet (HFD). MethodsBilateral ovariectomized female mice were fed an HFD for 6 weeks, and treated them with G-1. A cardiomyocyte insulin resistance model was used to simulate the in vivo environment. The main outcome measures were blood glucose, body weight, and serum insulin levels to assess insulin resistance, while cardiac function and degree of fibrosis were assessed by cardiac ultrasound and pathological observations. We also examined the expression of p-AMPK, p-AKT, and GLUT4 in mice hearts and in vitro models to explore the mechanism by which G-1 regulates insulin signaling. ResultsG-1 reduced body weight in mice on an HFD, but simultaneously increased blood glucose and promoted insulin resistance, resulting in myocardial damage. This damage included disordered cardiomyocytes, massive accumulation of glycogen, extensive fibrosis of the heart, and thickening of the front and rear walls of the left ventricle. At the molecular level, G-1 enhances gluconeogenesis and promotes glucose production by increasing the activity of pyruvate carboxylase (PC) while inhibiting GLUT4 translocation via the AMPK/TBC1D1 pathway, thereby limiting glucose uptake. ConclusionDespite G-1's the potential efficacy in weight reduction, the concomitant induction of insulin resistance and cardiac impairment in conjunction with an HFD raises significant concerns. Therefore, comprehensive studies of its safety profile and effects under specific conditions are essential prior to clinical use.

Protein targeting to glycogen is a master regulator of glycogen synthesis in astrocytes

The storage and use of glycogen, the main energy reserve in the brain, is a metabolic feature of astrocytes. Glycogen synthesis is regulated by Protein Targeting to Glycogen (PTG), a member of specific glycogen-binding subunits of protein phosphatase-1 (PPP1). It positively regulates glycogen synthesis through de-phosphorylation of both glycogen synthase (activation) and glycogen phosphorylase (inactivation). In cultured astrocytes, PTG mRNA levels were previously shown to be enhanced by the neurotransmitter noradrenaline. To achieve further insight into the role of PTG in the regulation of astrocytic glycogen, its levels of expression were manipulated in primary cultures of mouse cortical astrocytes using adenovirus-mediated overexpression of tagged-PTG or siRNA to downregulate its expression. Infection of astrocytes with adenovirus led to a strong increase in PTG expression and was associated with massive glycogen accumulation (>100 fold), demonstrating that increased PTG expression is sufficient to induce glycogen synthesis and accumulation. In contrast, siRNA-mediated downregulation of PTG resulted in a 2-fold decrease in glycogen levels. Interestingly, PTG downregulation strongly impaired long-term astrocytic glycogen synthesis induced by insulin or noradrenaline. Finally, these effects of PTG downregulation on glycogen metabolism could also be observed in cultured astrocytes isolated from PTG-KO mice. Collectively, these observations point to a major role of PTG in the regulation of glycogen synthesis in astrocytes and indicate that conditions leading to changes in PTG expression will directly impact glycogen levels in this cell type.

Mouse model of glycogen storage disease type III

Glycogen storage disease type IIIa (GSD IIIa) is caused by a deficiency of the glycogen debranching enzyme (GDE), which is encoded by the Agl gene. GDE deficiency leads to the pathogenic accumulation of phosphorylase limit dextrin (PLD), an abnormal glycogen, in the liver, heart, and skeletal muscle. To further investigate the pathological mechanisms behind this disease and develop novel therapies to treat this disease, we generated a GDE-deficient mouse model by removing exons after exon 5 in the Agl gene. GDE reduction was confirmed by western blot and enzymatic activity assay. Histology revealed massive glycogen accumulation in the liver, muscle, and heart of the homozygous affected mice. Interestingly, we did not find any differences in the general appearance, growth rate, and life span between the wild-type, heterozygous, and homozygous affected mice with ad libitum feeding, except reduced motor activity after 50weeks of age, and muscle weakness in both the forelimb and hind legs of homozygous affected mice by using the grip strength test at 62weeks of age. However, repeated fasting resulted in decreased survival of the knockout mice. Hepatomegaly and progressive liver fibrosis were also found in the homozygous affected mice. Blood chemistry revealed that alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities were significantly higher in the homozygous affected mice than in both wild-type and heterozygous mice and the activity of these enzymes further increased with fasting. Creatine phosphokinase (CPK) activity was normal in young and adult homozygous affected mice. However, the activity was significantly elevated after fasting. Hypoglycemia appeared only at a young age (3weeks) and hyperlipidemia was not observed in our model. In conclusion, with the exception of normal lipidemia, these mice recapitulate human GSD IIIa; moreover, we found that repeated fasting was detrimental to these mice. This mouse model will be useful for future investigation regarding the pathophysiology and treatment strategy of human GSD III.

Gingival Overgrowth in Pompe Disease: A Case Report

Pompe disease, or glycogen storage disease type 2, is a rare inheritable metabolic disease caused by a deficiency of the lysosomal enzyme acid α-glucosidase. Patients with the classic infantile form of Pompe disease present with symptoms during the first 3 months after birth, and most will die within their first year. Recently, enzyme replacement therapy (ERT) with recombinant human α-glucosidase became commercially available for Pompe disease. This is a case report of an 8-year-old girl with the infantile form of Pompe disease who is one of the longest survivors through ERT. The patient was tetraplegic when she started ERT. At age 3 years, she developed massive gingival overgrowth and could not close her mouth, prompting a reduction of the gingival overgrowth surgically. We expected that massive accumulation of glycogen would explain the gingival overgrowth. However, histopathology of the gingiva tissue showed marked glycogen accumulation in smooth muscle cells of the arteries, but the glycogen content in fibroblasts did not exceed that of control individuals. Further, there was an increase of immature collagen in the connective tissue, and signs of a mild chronic inflammation. We concluded that glycogen storage is not a direct causative factor of gingival overgrowth in our patient. Chronic inflammation, dryness of the gingiva, or even the minimal glycogen accumulation in the fibroblasts may have played a role.

Evaluation of Systemic Follistatin as an Adjuvant to Stimulate Muscle Repair and Improve Motor Function in Pompe Mice

Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.

Activation of glycolysis and apoptosis in glycogen storage disease type Ia

The deficiency of glucose-6-phosphatase (G6Pase) underlies glycogen storage disease type Ia (GSD-Ia, von Gierke disease; MIM 232200), an autosomal recessive disorder of metabolism associated with life-threatening hypoglycemia, growth retardation, renal failure, hepatic adenomas, and hepatocellular carcinoma. Liver involvement includes the massive accumulation of glycogen and lipids due to accumulated glucose-6-phosphate and glycolytic intermediates. Proteomic analysis revealed elevations in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and other enzymes involved in glycolysis. GAPDH was markedly increased in murine G6Pase-deficient hepatocytes. The moonlighting role of GAPDH includes increasing apoptosis, which was demonstrated by increased TUNEL assay positivity and caspase 3 activation in the murine GSD-Ia liver. These analyses of hepatic involvement in GSD-Ia mice have implicated the induction of apoptosis in the pathobiology of GSD-Ia.

Vascular Endothelial Growth Factor and Nitric Oxide Production in Response to Hypoxia in the Choroid Plexus in Neonatal Brain

Damage to the choroid plexus in 1-day-old Wistar rats subjected to hypoxia was investigated. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal, inducible nitric oxide synthase (eNOS, nNOS, iNOS), and vascular endothelial growth factor (VEGF) along with nitric oxide (NO) production and VEGF concentration was up-regulated significantly in hypoxic rats. Ultrastructurally, the choroid plexus epithelial cells showed massive accumulation of glycogen. A striking feature was the extrusion of cytoplasmic fragments from the apical cell surfaces into the ventricular lumen following the hypoxic insult. Intraventricular macrophages showed increased expression of complement type 3 receptors, major histocompatibility complex class I and II antigens, and ED1 antigens. Following an intravenous injection of horseradish peroxidase (HRP), a large number of intraventricular macrophages were labeled suggesting enhanced leakage of the tracer from the blood vessels in the choroid plexus connective tissue stroma into the ventricular lumen. We suggest that increased production of NO in hypoxia is linked to the structural alteration of the choroid plexus, and along with VEGF, may lead to increased vascular permeability. Melatonin treatment reduced VEGF and NO levels as well as leakage of HRP suggesting its potential value in ameliorating damage in choroid plexus pathologies.

Adaptive changes in interscapular brown adipose tissue during reacclimation after cold: The role of redox regulation

1. The level of antioxidative defence, intensity of sympathetic innervation, expression of uncoupling protein 1 (UCP1), and ultrastructure of interscapular brown adipose tissue (IBAT) were studied in rats acclimated to room (22±1 °C), low temperature (4±1 °C, 45 days) and during reacclimation to 22 °C (days 1, 3, and 7). 2. The cold-induced increase in catalase, glutathione peroxidase, glutathione reductase, thioredoxin reductase, as well as a decrease in the activities of CuZn- and Mn-superoxide dismutases, shows specific dynamics of restitution during reacclimation to 22 °C. 3. The intensity of the cold-induced enhancement of the sympathetic innervation and UCP1 expression gradually decreases during reacclimation to 22 °C, but with the seventh day level still higher compared to the animals acclimated to room temperature. 4. Electron-microscopy study of IBAT shows that changes of the strongly activated tissue in cold undergo gradual restitution during reacclimation to 22 °C, but are observed already on the first day through a massive accumulation of glycogen, accompanied by a change in the lipid profile.

33. Systemic Delivery of AAV8/DC190-GAA to Pompe Mice Normalizes Muscle Pathology and Corrects Motor Function Deficits

Pompe disease, a lysosomal storage disorder, is generally known for it's characteristic degenerative myopathy. Massive accumulation of glycogen in lysosomes of striated and smooth muscle due to a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) results in muscle weakness and cardiorespiratory failure. In severely affected patients glycogen also accumulates in the central and peripheral nervous systems. Previously we demonstrated that systemic administration of AAV8/DC190-GAA to Pompe mice generated high circulating levels of GAA and reduced glycogen storage in cardiac and skeletal muscle to near normal levels. The current study was designed to determine if these effects on the viscera would be sufficient to prevent the loss of muscle strength and coordination shown to develop over time in the Pompe mouse. Since the GAA secreted into the blood should not cross the bloodbrain bloodbrain barrier, this study should also clarify the functional significance of peripheral and CNS glycogen storage in the Pompe mouse model. Two different concentrations of AAV8/DC190-GAA were injected into the tail vein of 10-week-old male Pompe mice. Treated Pompe mice, as well as vehicle-treated Pompe and B6129SF2/J normal control mice, were tested for their ability to perform in a battery of behavioral tests. Tests designed to measure muscle strength and coordination included accelerating and rocking Rota-rod, wire hang, and foot fault. As expected, the performance of the vehicle-treated Pompe mice declined over time when compared to wild type mice. In contrast the performance of AAV-treated Pompe mice was significantly improved over that of vehicle-treated Pompe mice, and was similar to normal mice. There was no significant difference between the two AAV8 dosing groups in any test except the wire hang. In this test the performance of the lower dose treatment group declined at the later time points. Severely affected patients rapidly accumulate glycogen, and infants are often not diagnosed until they show obvious myotonia. To model the treatment of this patient population, we administered AAV8/DC190-GAA to 10-month-old Pompe mice, an age at which they display significant muscle pathology and motor function decline. Mice treated at this advanced stage of the disease showed a partial recovery of muscle function, but performance was not normalized despite high circulating levels of GAA. We are currently measuring glycogen levels and studying the histopathology of muscle and CNS tissues from the mice in these studies. In summary, we have shown that systemic gene therapy treatment is capable of preventing degenerative myopathy and preserving motor function in Pompe mice, but is less effective in animals with pre-existing pathology.

1124. Correction of Glycogen Storage Disease Type II by an AAV Vector Encoding Highly-Secreted, Chimeric Acid α-glucosidase

Top of pageAbstract GSD-II (Pompe disease; MIM 232300) is an autosomal recessive disorder caused by the deficiency in the activity of the lysosomal enzyme acid |[alpha]|-glucosidase (GAA; acid maltase; EC 3.2.1.20). The disease is characterized by the massive accumulation of glycogen in lysosomes and in the cytoplasm of striated muscle, with an accompanying disruption of cellular functions. The efficacy of therapy with lysosomal enzymes, such as GAA, could be markedly improved if secretion of these proteins from producer cells were increased. Secreted proteins are directed extracellularly by the presence of an N-terminal signal peptide. We have investigated the hypothesis that a chimeric lysosomal enzyme containing an alternative signal peptide will achieve much higher secretion of the recombinant lysosomal enzyme from transduced cells. The coding sequence of human GAA was modified by substituting alternative signal peptides from highly secreted proteins for the native GAA signal peptide (denoted chimeric GAA). The relative secretion of chimeric GAA from transfected 293 cells increased up to 26-fold, compared to the secretion of unmodified GAA. The efficacy of this strategy for gene therapy in Pompe disease was analyzed in GAA-knockout (GAA-KO) mice. An adeno-associated virus (AAV) vector containing a liver-specific promoter to drive the expression of GAA was constructed (AAV-LSPSPhGAApA) and pseudotyped as AAV8. The long-term efficacy of SP-hGAA was evaluated by the quantitation of GAA activity and glycogen content following AAV-LSPSPhGAApA administration. A dose-response analysis revealed sustained, high GAA activity in plasma following administration of at least 3 |[times]| 1010 particles; however, an equivalent number of particles for a vector encoding unmodified GAA did not elevate GAA activity in plasma. Receptor-mediated uptake of secreted GAA can be demonstrated only if plasma levels exceed a therapeutic threshold, and the GAA activity of the heart and diaphragm were significantly elevated 18 weeks following administration of as few as 1 |[times]| 1010 particles of AAV-LSPSPhGAApA. GAA activity in the quadriceps muscle was increased with as few as 3 |[times]| 1010 particles of AAV-LSPSPhGAApA. The lower threshold for significant reduction of heart, diaphragm, and quadriceps glycogen content with AAV-LSPSPhGAApA was 3 |[times]| 1010 particles. Neither fewer particles for that vector, nor an equivalent number of particles of the analogous vector encoding unmodified GAA affected glycogen content in the quadriceps. Thus, an AAV vector encoding chimeric GAA increased the secretion of hGAA from the liver, and reduced the number of vector particles required to achieve a therapeutic effect in GSD-II mice.

Glycogen storage in multiple muscles of old GSD-II mice can be rapidly cleared after a single intravenous injection with a modified adenoviral vector expressing hGAA

Glycogen storage disease II (GSD-II) is an autosomal recessive lysosomal storage disease, due to acid-alpha-glucosidase (GAA) deficiency. The disease is characterized by massive glycogen accumulation in the cardiac and skeletal muscles. There is early onset (infantile, also known as Pompe disease) as well as late onset (juvenile and adult) forms of GSD-II. Few studies have been published to date that have explored the consequences of delivering a potential therapy to either late onset GSD-II subjects, and/or early onset patients with long-established muscle pathology. One recent report utilizing GAA-KO mice transgenically expressing human GAA (hGAA) suggested that long-established disease in both cardiac and skeletal muscle is likely to prove resistant to therapies. To investigate the potential for disease reversibility in old GSD-II mice, we studied their responsiveness to exogenous hGAA exposure via a gene therapy approach that we have previously shown to be efficacious in young GAA-KO mice. An [E1-, polymerase-] adenoviral vector encoding hGAA was intravenously injected into two groups of aged GAA-KO mice; GAA expression and tissue glycogen reduction were evaluated. After vector injection, we found that extremely high amounts of hepatically secreted hGAA could be produced, and subsequently taken up by multiple muscle tissues in the old GAA-KO mice by 17 days post-injection (dpi). As a result, all muscle groups tested in the old GAA-KO mice showed significant glycogen reductions by 17 dpi, relative to that of age-matched, but mock-injected GAA-KO mice. For example, glycogen reduction in heart was 84%, in quadriceps 46%, and in diaphragm 73%. Our data also showed that the uptake and the subsequent intracellular processing of virally expressed hGAA were not impaired in older muscles. Overall, the previously reported 'resistance' of old GAA-KO muscles to exogenous hGAA replacement approaches can be rapidly overcome after a single intravenous injection with a modified adenoviral vector expressing hGAA.

699. Correction of Glycogen Storage Disease Type II (GSD II) with an Adeno-Associated Virus 8 (AAV2/8) Vector

Glycogen storage disease type II (GSD II; Pompe disease; MIM 232300) is an autosomal recessive disorder caused by a deficiency in the activity of the lysosomal enzyme alpha-glucosidase (GAA; acid maltase; EC 3.2.1.20). The disease is characterized by massive accumulation of glycogen in the lysosomes and cytoplasm of striated muscle, with a subsequent disruption of cellular functions. Clinically, the disease spectrum varies in severity and age of onset, and the manifestations include muscle weakness, hypotonia, hypertrophic cardiomyopathy and respiratory failure. Intravenous administration of adenovirus vectors encoding GAA previously demonstrated generalized correction of glycogen storage in the GAA-knockout (GAA-KO) mouse model, although GAA secretion stopped and glycogen content increased coincident with appearance of anti-GAA antibodies. Tissue-specific expression of an introduced therapeutic protein has prevented the formation of neutralizing antibodies in other models, but not in GAA-KO mice. Consequently, an AAV2/8 vector containing a liver-specific promoter (LSP) to drive GAA expression (AAV-LSPGAApA) was administered (1 x 10E11 particles intravenously) in 3 month-old immunocompetent GAA-KO mice. A second group of age-matched GAA-KO mice was injected with a similar dose of an AAV2/8 vector containing hGAA under the control of the CMV enhancer/chicken |[beta]|-actin (CB) promoter for comparison (AAV-CBGAApA). High-level hGAA was detectable by Western blot in plasma for longer than 6 weeks with AAV-LSPGAApA in GAA-KO mice, in contrast to AAV-CBGAApA which produced low-level hGAA in the plasma only during the first week. The latter vector has produced sustained, high-level hGAA in plasma in immunodeficient (GAA-KO/SCID) mice, indicating the significance of the immune response to hGAA secretion. ELISA performed at 1, 3, and 6 weeks following vector administration demonstrated no detectable anti-GAA antibodies in response to liver-specific hGAA expression in GAA-KO mice ( 1:800) at the 6 week time point. The level of hGAA activity in plasma with AAV-LSPGAApA (>1000 nmol/hr/ml) has previously corrected glycogen storage in the heart of tolerant GAA-KO mice. This combined approach to liver-targeted gene therapy of GSD II with a liver-directed AAV serotype and a liver-specific promoter provides a substantially greater and more sustained level of hGAA expression in vivo than has previously been reported. Further work will be carried out to determine the implications of this sustained, high-level expression of the enzyme on disease phenotype. Thus, use of a liver-specific promoter prevented neutralizing antibody formation in GSD II mice and will enhance the development of gene therapy in Pompe disease.

862. Improved Efficacy of Adenovirus-Mediated Gene Therapy for GSD-II Disease by Selective Depletion of Kupffer Cells

Glycogen storage disease type II (GSD-II) is a fatal genetic muscle disorder caused by a massive accumulation of glycogen in multiple muscle groups, and is caused by the deficiency of intralysosomal acid alpha glucosidase (GAA). Two approaches to the treatment of GSD-II are currently being pursued in our research group: enzyme replacement therapy and gene therapy. Relative to gene therapy, our previous work demonstrated that a single intravenous administration of advanced generation, [E1-,pol-] adenoviral (Ad) based vectors encoding human GAA (hGAA) resulted in efficient hepatic transduction and secretion of high levels of hGAA, consequently, the correction of all affected muscles in a GAA-KO mouse model as well a quail model of GSD-II occurred. The long-term persistence of the improved vector in liver tissues facilitated significant glycogen reduction in multiple muscles (though, with variable responses in extent and duration among the individual muscle group analyzed) for at least 6 months, despite the fact that hGAA is perceived as a foreign antigen in these animal models. Unfortunately, our most recent work suggests that very high doses of Ad vectors must be intravenously injected to facilitate the large amounts of hepatic hGAA secretion required to correct both cardiac and skeletal muscles in these animal models. To determine if this problem can be overcome, we have begun attempts to pre-emptively suppress the innate immune systems of immunocompetent GAA-KO mice, prior to injection with our viral vectors. Macrophages, in particular hepatic Kupffer cells, play a central role in sequestering Ad vectors, as well in triggering early innate immune responses, and hence have a profound impact on the adaptive immune responses following adenoviral vector administration. In this study we evaluated the effect of selective and transient depletion of Kupffer cells (via pretreatment of mice with clodronate liposomes-dichloromethylene-bisphosphonate) one day prior to vector injection. We intravenously injected into the clodronate liposome pretreated GAA-KO mice different dosages of the vector, to determine how removal of Kupffer cells impacted upon the known efficacy of this approach. We found that clodronate pretreatment allowed lower doses of the vector to show greater efficacy. For example, the GAA activities in the clodronate-treated mice were significantly elevated both in plasma and in tissues such as liver, heart, diaphram and quadriceps, as compared to the non-Kupffer cell depleted mice injected with identical doses of the [E1-,pol-]AdhGAA vector. As a result, the pathogenic glycogen accumulations in clodronate liposome pretreated GAA-KO mice were significantly reduced at lower doses of the Ad vector (10–100 fold less) as compared to Ad treated GAA-KO mice that had not been pretreated with clodronate containing liposomes. Our data also suggests however, that liver toxicities were also now apparent at lower vector dosages, suggesting that Kupffer cells act to prevent liver damage by sequestering Ad particles. Whether or not other “Ad induced” toxicities are prevented by Kupffer cell depletion are studies currently ongoing in our laboratories.

Helios gene gun particle delivery for therapy of acid maltase deficiency.

Autosomal recessive deficiency of lysosomal acid maltase (GAA) or glycogen storage disease type II (GSDII) results in a spectrum of phenotypes including a rapidly fatal infantile disorder (Pompe's), juvenile, and a late-onset adult myopathy. The infantile onset form presents as hypotonia with massive accumulation of glycogen in skeletal and heart muscle, with death due to cardiorespiratory failure. Adult patients with the slowly progressive form develop severe skeletal muscle weakness and respiratory failure. Particle bombardment is a safe, efficient physical method in which high-density, subcellular-sized particles are accelerated to high velocity to carry DNA into cells. Because it does not depend on a specific ligand, receptor, or biochemical features on cell surfaces, particle-mediated gene transfer can be readily applied to a variety of systems. We evaluated particle bombardment as a delivery system for therapy of GSDII. We utilized a vector carrying the CMV promoter linked to the human GAA cDNA. Human GSDII cell lines (fibroblasts and lymphoid) as well as ex vivo with adult-onset peripheral blood cells (lymphocytes and monocytes) were transiently transfected by bombardment with a Helios gene gun delivering gold particles coated with the GAA expression plasmid. All cell types showed an increase in human GAA activity greater than 50% of normal activity. Subsequently, GAA -/- mice were treated every 2 weeks for 4 months by particle bombardment to the epidermis of the lower back and hind limbs. Muscle weakness in the hind and forelimbs was reversed. These data suggest that particle delivery of the GAA cDNA by the Helios gene gun may be a safe, effective treatment for GSDII.

Acute Inhibition of Hepatic Glucose-6-phosphatase Does Not Affect Gluconeogenesis but Directs Gluconeogenic Flux toward Glycogen in Fasted Rats: A PHARMACOLOGICAL STUDY WITH THE CHLOROGENIC ACID DERIVATIVE S4048

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.

Glycogen accumulation in the ovarian follicle of Indian vespertilionid bat, Scotophilus heathi during the period of delayed ovulation.

Massive accumulation of glycogen in the follicles of Scotophilus heathi during the period of delayed ovulation was noticed. The follicles which survive for prolonged periods are morphologically specialized. The accumulation of glycogen was noticed in the granulosa cells. The egg cytoplasm, thecal cells and interstitial cells were almost devoid of glycogen. The first sign of a glycogen deposit in the ovary was noticed during the recrudescence phase (October) in late pre-antral follicles. Most of the morphologically healthy late pre-antral and antral follicles showed a positive reaction with PAS and Best carmine from October to early February. Morphologically atretic follicles showed only a mild glycogen accumulation. Little or no glycogen accumulation was noticed in some of the healthy late-antral follicles before ovulation during late February and early March. HCG-induced antral follicles during quiescence also did not show any accumulation of glycogen. The study suggests that glycogen laden follicles are not suitable for ovulation and may be the reason for the occurrence of delayed ovulation in S. heathi.

Glycogen storage disease with normal acid maltase: Skeletal and cardiac muscles

We report a 5-year-old boy with lysosomal glycogen storage disease and normal acid maltase activity. This patient, the fourth reported in the literature, was referred to our hospital for evaluation of elevated serum GOT, GPT, and CK activities. He had neither muscle weakness nor atrophy. Echocardiography demonstrated marked thickening of the intraventricular septum and left ventricular wall which indicated hypertrophic cardiomyopathy. Biopsied skeletal muscle disclosed massive accumulation of glycogen and autophagic vacuoles. Electron microscopy of biopsied cardiac muscle revealed severe myofibrillar disruption with marked accumulation of free and intralysosomal glycogen. Activities of all major glycolytic enzymes in skeletal muscle, including acid maltase, were normal. It is unknown why muscle lysosomes appeared to be unable to digest the trapped glycogen despite the presence of acid maltase. Our findings illustrate the importance of performing skeletal muscle investigation during childhood in patients with hypertrophic cardiomyopathy.

Prepartum and postpartum glycogen accumulation in bovine uterine arteries

Histochemical examination of bovine uterine arteries revealed that massive accumulation of glycogen occurred in the tunica media of the arteries at the late gestational and early puerperal stages. The amount of glycogen appeared to increase progressively during pregnancy and then decreased gradually after parturition. In contrast, little glycogen was found in cycling cows. There was no direct relationship between glycogen deposition and frequency of parturition. These results suggest that massive storage of glycogen in the uterine artery may serve as an important source of energy for the recovery of the uterus and its arteries to their nonpregnant states.

Nitrogen and Sulfur Starvation of the Cyanobacterium Synechococcus 6301 An Ultrastructural, Morphometrical, and Biochemical Comparison

Abstract The effects of nitrogen and sulfur limitation on various cellular parameters of the cyanobacterium Synechococcus 6301 were studied by electron microscopy, morphometry and biochemical methods. Nitrate and sulfate starvation for 70 h results in a massive glycogen accumulation in parallel to a loss of soluble protein and chlorophyll. Phycobilisomes disappear prior to the degradation of photosynthetic membranes. For sulfate-starved cells, a formation of “storage granules” (poly-β-hydroxy-butyric acid) is typical which amount up to 10% of the cell volume. The composition of polar lipids is simple: equal parts of C 16:0 and C 16:1 are present under all nutritional conditions; their amount is directly correlated with the total cellular membrane area as deter­mined by morphometry. Nitrate starved cells regenerate almost completely in structure and composition within 9 h after nitrate supplementation. Regeneration of sulfate starved cells is retarded; in spite of significant synthesis of phycocyanin within 9 h the cells still exhibit marked signs of starvation.

Galactose neuropathy: impact of chronic endoneurial edema on nerve blood flow.

Peripheral neuropathy induced by galactose feeding results in endoneurial edema with increased tissue pressure and ultimate demyelination of nerve fibers. To assess the role of ischemia in the pathogenesis of this neuropathy, nerve blood flow was measured 6 months after the start of galactose ingestion, between the onset of edema and nerve fiber changes. Measurement was carried out by a noninvasive technique involving the use of [14C]iodoantipyrine as a radioactive tracer of tissue perfusion. A significant decline in nerve blood flow was found, which correlated positively with increased nerve water content as quantified by a microgravimetric technique. We sought to establish the pathogenic role of nerve edema in the development of schwannopathy and demyelination by morphological examination. It was found that Schwann cells appeared normal in areas of the peripheral nervous system in which edema does not occur, such as spinal ganglia and roots, whereas in regions in which there was edema, massive glycogen accumulation in Schwann cells and demyelination occurred. These findings suggest that edema, rather than hyperactivity in the sorbitol pathway, is responsible for the pathological changes in galactosemic neuropathy and that ischemia resulting from edema and increased endoneurial fluid pressure is the mechanism responsible for fiber injury.