Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation.

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10-12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.

Model-based analysis uncovers mutations altering autophagy selectivity in human cancer.

Autophagy can selectively target protein aggregates, pathogens, and dysfunctional organelles for the lysosomal degradation. Aberrant regulation of autophagy promotes tumorigenesis, while it is far less clear whether and how tumor-specific alterations result in autophagic aberrance. To form a link between aberrant autophagy selectivity and human cancer, we establish a computational pipeline and prioritize 222 potential LIR (LC3-interacting region) motif-associated mutations (LAMs) in 148 proteins. We validate LAMs in multiple proteins including ATG4B, STBD1, EHMT2 and BRAF that impair their interactions with LC3 and autophagy activities. Using a combination of transcriptomic, metabolomic and additional experimental assays, we show that STBD1, a poorly-characterized protein, inhibits tumor growth via modulating glycogen autophagy, while a patient-derived W203C mutation on LIR abolishes its cancer inhibitory function. This work suggests that altered autophagy selectivity is a frequently-used mechanism by cancer cells to survive during various stresses, and provides a framework to discover additional autophagy-related pathways that influence carcinogenesis.

Three-dimensional periodontal tissue regeneration using a bone-ligament complex cell sheet.

Periodontal tissue is a distinctive tissue structure composed three-dimensionally of cementum, periodontal ligament (PDL) and alveolar bone. Severe periodontal diseases cause fundamental problems for oral function and general health, and conventional dental treatments are insufficient for healing to healthy periodontal tissue. Cell sheet technology has been used in many tissue regenerations, including periodontal tissue, to transplant appropriate stem/progenitor cells for tissue regeneration of a target site as a uniform tissue. However, it is still difficult to construct a three-dimensional structure of complex tissue composed of multiple types of cells, and the transplantation of a single cell sheet cannot sufficiently regenerate a large-scale tissue injury. Here, we fabricated a three-dimensional complex cell sheet composed of a bone-ligament structure by layering PDL cells and osteoblast-like cells on a temperature responsive culture dish. Following ectopic and orthotopic transplantation, only the complex cell sheet group was demonstrated to anatomically regenerate the bone-ligament structure along with the functional connection of PDL-like fibers to the tooth root and alveolar bone. This study represents successful three-dimensional tissue regeneration of a large-scale tissue injury using a bioengineered tissue designed to simulate the anatomical structure.

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic ß-cells.

Diabetes is a global health problem caused primarily by the inability of pancreatic ß-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of ß-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic ßV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 ß-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in ß-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of ß-cells in diabetes.

Laforin-malin complex degrades polyglucosan bodies in concert with glycogen debranching enzyme and brain isoform glycogen phosphorylase.

In Lafora disease (LD), the deficiency of either EPM2A or NHLRC1, the genes encoding the phosphatase laforin and E3 ligase, respectively, causes massive accumulation of less-branched glycogen inclusions, known as Lafora bodies, also called polyglucosan bodies (PBs), in several types of cells including neurons. The biochemical mechanism underlying the PB accumulation, however, remains undefined. We recently demonstrated that laforin is a phosphatase of muscle glycogen synthase (GS1) in PBs, and that laforin recruits malin, together reducing PBs. We show here that accomplishment of PB degradation requires a protein assembly consisting of at least four key enzymes: laforin and malin in a complex, and the glycogenolytic enzymes, glycogen debranching enzyme 1 (AGL1) and brain isoform glycogen phosphorylase (GPBB). Once GS1-synthesized polyglucosan accumulates into PBs, laforin recruits malin to the PBs where laforin dephosphorylates, and malin degrades the GS1 in concert with GPBB and AGL1, resulting in a breakdown of polyglucosan. Without fountional laforin-malin complex assembled on PBs, GPBB and AGL1 together are unable to efficiently breakdown polyglucosan. All these events take place on PBs and in cytoplasm. Deficiency of each of the four enzymes causes PB accumulation in the cytoplasm of affected cells. Demonstration of the molecular mechanisms underlying PB degradation lays a substantial biochemical foundation that may lead to understanding how PB metabolizes and why mutations of either EPM2A or NHLRC1 in humans cause LD. Mutations in AGL1 or GPBB may cause diseases related to PB accumulation.

An optimized histochemical method to assess skeletal muscle glycogen and lipid stores reveals two metabolically distinct populations of type I muscle fibers.

Skeletal muscle energy metabolism has been a research focus of physiologists for more than a century. Yet, how the use of intramuscular carbohydrate and lipid energy stores are coordinated during different types of exercise remains a subject of debate. Controversy arises from contradicting data from numerous studies, which used different methodological approaches. Here we review the "pros and cons" of previously used histochemical methods and describe an optimized method to ensure the preservation and specificity of detection of both intramyocellular carbohydrate and lipid stores. For optimal preservation of muscle energy stores, air drying cryosections or cycles of freezing-thawing need to be avoided. Furthermore, optimization of the imaging settings in order to specifically image intracellular lipid droplets stained with oil red O or Bodipy-493/503 is shown. When co-staining lipid droplets with associated proteins, Bodipy-493/503 should be the dye of choice, since oil red O creates precipitates on the lipid droplets blocking the light. In order to increase the specificity of glycogen stain, an antibody against glycogen is used. The resulting method reveals the existence of two metabolically distinct myosin heavy chain I expressing fibers: I-1 fibers have a smaller crossectional area, a higher density of lipid droplets, and a tendency to lower glycogen content compared to I-2 fibers. Type I-2 fibers have similar lipid content than IIA. Exhaustive exercise lead to glycogen depletion in type IIA and IIX fibers, a reduction in lipid droplets density in both type I-1 and I-2 fibers, and a decrease in the size of lipid droplets exclusively in type I-1 fibers.

Polyglucosan neurotoxicity caused by glycogen branching enzyme deficiency can be reversed by inhibition of glycogen synthase.

Uncontrolled elongation of glycogen chains, not adequately balanced by their branching, leads to the formation of an insoluble, presumably neurotoxic, form of glycogen called polyglucosan. To test the suspected pathogenicity of polyglucosans in neurological glycogenoses, we have modeled the typical glycogenosis Adult Polyglucosan Body Disease (APBD) by suppressing glycogen branching enzyme 1 (GBE1, EC 2.4.1.18) expression using lentiviruses harboring short hairpin RNA (shRNA). GBE1 suppression in embryonic cortical neurons led to polyglucosan accumulation and associated apoptosis, which were reversible by rapamycin or starvation treatments. Further analysis revealed that rapamycin and starvation led to phosphorylation and inactivation of glycogen synthase (GS, EC 2.4.1.11), dephosphorylated and activated in the GBE1-suppressed neurons. These protective effects of rapamycin and starvation were reversed by overexpression of phosphorylation site mutant GS only if its glycogen binding site was intact. While rapamycin and starvation induce autophagy, autophagic maturation was not required for their corrective effects, which prevailed even if autophagic flux was inhibited by vinblastine. Furthermore, polyglucosans were not observed in any compartment along the autophagic pathway. Our data suggest that glycogen branching enzyme repression in glycogenoses can cause pathogenic polyglucosan buildup, which might be corrected by GS inhibition.

Laforin prevents stress-induced polyglucosan body formation and Lafora disease progression in neurons.

Glycogen, the largest cytosolic macromolecule, is soluble because of intricate construction generating perfect hydrophilic-surfaced spheres. Little is known about neuronal glycogen function and metabolism, though progress is accruing through the neurodegenerative epilepsy Lafora disease (LD) proteins laforin and malin. Neurons in LD exhibit Lafora bodies (LBs), large accumulations of malconstructed insoluble glycogen (polyglucosans). We demonstrated that the laforin-malin complex reduces LBs and protects neuronal cells against endoplasmic reticulum stress-induced apoptosis. We now show that stress induces polyglucosan formation in normal neurons in culture and in the brain. This is mediated by increased glucose-6-phosphate allosterically hyperactivating muscle glycogen synthase (GS1) and is followed by activation of the glycogen digesting enzyme glycogen phosphorylase. In the absence of laforin, stress-induced polyglucosans are undigested and accumulate into massive LBs, and in laforin-deficient mice, stress drastically accelerates LB accumulation and LD. The mechanism through which laforin-malin mediates polyglucosan degradation remains unclear but involves GS1 dephosphorylation by laforin. Our work uncovers the presence of rapid polyglucosan metabolism as part of the normal physiology of neuroprotection. We propose that deficiency in the degradative phase of this metabolism, leading to LB accumulation and resultant seizure predisposition and neurodegeneration, underlies LD.

Visualization of the superior and inferior borders of the mandibular canal: a comparative study using digital panoramic radiographs and cross-sectional computed tomography images.

OBJECTIVE: To evaluate the visibility of the superior and inferior borders of mandibular canal using panoramic radiography (PR) and cross-sectional computed tomography (CT) images. STUDY DESIGN: Digital panoramic images and cross-sectional CT images of 100 patients were evaluated. The mandibular canal was divided into 4 areas of equal width (1-4), from anterior to posterior. The visibility of the superior and inferior borders was assessed using a 5-point visibility scoring system, with lower scores for worse visibility. RESULTS: For both modalities, the superior border showed significantly lower score than the inferior border in all areas. For the superior border, areas 1, 2, and 3 all showed significantly lower scores than area 4 for PR, whereas only area 1 showed a lower score than area 4 for CT. CONCLUSIONS: The visibility of the superior border was very poor on panoramic images. The use of cross-sectional CT images remarkably improved this poor visualization.

Laforin is required for the functional activation of malin in endoplasmic reticulum stress resistance in neuronal cells.

Mutations in either EPM2A, the gene encoding a dual-specificity phosphatase named laforin, or NHLRC1, the gene encoding an E3 ubiquitin ligase named malin, cause Lafora disease in humans. Lafora disease is a fatal neurological disorder characterized by progressive myoclonus epilepsy, severe neurological deterioration and accumulation of poorly branched glycogen inclusions, called Lafora bodies or polyglucosan bodies, within the cell cytoplasm. The molecular mechanism underlying the neuropathogenesis of Lafora disease remains unknown. Here, we present data demonstrating that in the cells expressing low levels of laforin protein, overexpressed malin and its Lafora disease-causing missense mutants are stably polyubiquitinated. Malin and malin mutants form ubiquitin-positive aggregates in or around the nuclei of the cells in which they are expressed. Neither wild-type malin nor its mutants elicit endoplasmic reticulum stress, although the mutants exaggerate the response to endoplasmic reticulum stress. Overexpressed laforin impairs the polyubiquitination of malin while it recruits malin to polyglucosan bodies. The recruitment and activities of laforin and malin are both required for the polyglucosan body disruption. Consistently, targeted deletion of laforin in brain cells from Epm2a knockout mice increases polyubiquitinated proteins. Knockdown of Epm2a or Nhlrc1 in neuronal Neuro2a cells shows that they cooperate to allow cells to resist ER stress and apoptosis. These results reveal that a functional laforin-malin complex plays a critical role in disrupting Lafora bodies and relieving ER stress, implying that a causative pathogenic mechanism underlies their deficiency in Lafora disease.

Glucose uptake mediated by glucose transporter 1 is essential for early tooth morphogenesis and size determination of murine molars.

Glucose is an essential source of energy for body metabolism and is transported into cells by glucose transporters (GLUTs). Well-characterized class I GLUT is subdivided into GLUTs1-4, which are selectively expressed depending on tissue glucose requirements. However, there is no available data on the role of GLUTs during tooth development. This study aims to clarify the functional significance of class I GLUT during murine tooth development using immunohistochemistry and an in vitro organ culture experiment with an inhibitor of GLUTs1/2, phloretin, and Glut1 and Glut2 short interfering RNA (siRNA). An intense GLUT1-immunoreaction was localized in the enamel organ of bud-stage molar tooth germs, where the active cell proliferation occurred. By the bell stage, the expression of GLUT1 in the dental epithelium was dramatically decreased in intensity, and subsequently began to appear in the stratum intermedium at the late bell stage. On the other hand, GLUT2-immunoreactivity was weakly observed in the whole tooth germs throughout all stages. The inhibition of GLUTs1/2 by phloretin in the bud-stage tooth germs induced the disturbance of primary enamel knot formation, resulting in the developmental arrest of the explants and the squamous metaplasia of dental epithelial cells. Furthermore, the inhibition of GLUTs1/2 in cap-to-bell-stage tooth germs reduced tooth size in a dose dependent manner. These findings suggest that the expression of GLUT1 and GLUT2 in the dental epithelial and mesenchymal cells seems to be precisely and spatiotemporally controlled, and the glucose uptake mediated by GLUT1 plays a crucial role in the early tooth morphogenesis and tooth size determination.

Histological demonstration of glucose transporters, fructose-1,6-bisphosphatase, and glycogen in gas gland cells of the swimbladder: is a metabolic futile cycle operating?

Luminal surface of the swimbladder is covered by gas gland epithelial cells and is responsible for inflating the swimbladder by generating O(2) from Root-effect hemoglobin that releases O(2) under acidic conditions. Acidification of blood is achieved by lactic acid secreted from gas gland cells, which are poor in mitochondria but rich in the glycolytic activity. The acidic conditions are locally maintained by a countercurrent capillary system called rete mirabile. To understand the regulation of anaerobic metabolism of glucose in the gas gland cells, we analyzed the glucose transporter expressed there and the fate of ATP generated by glycolysis. The latter is important because the ATP should be immediately consumed otherwise it strongly inhibits the glycolysis rendering the cells unable to produce lactic acid anymore. Expression analyses of glucose transporter (glut) genes in the swimbladder of fugu (Takifugu rubripes) by RT-PCR and in situ hybridization demonstrated that glut1a and glut6 are expressed in gas gland cells. Immunohistochemical analyses of metabolic enzymes demonstrated that a gluconeogenesis enzyme fructose-1,6-bisphosphatase (Fbp1) and a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (Gapdh) are highly expressed in gas gland cells. The simultaneous catalyses of glycolysis and gluconeogenesis reactions suggest the presence of a futile cycle in gas gland cells to maintain the levels of ATP low and to generate heat that helps reduce the solubility of O(2).

An immunohistochemical and ultrastructural study of the pericellular matrix of uneroded hypertrophic chondrocytes in the mandibular condyle of aged c-src-deficient mice.

OBJECTIVE: Previous studies indicate that hypertrophic chondrocytes can transdifferentiate or dedifferentiate and redifferentiate into bone cells during the endochondral bone formation. Mandibular condyle in aged c-src-deficient mice has incremental line-like striations consisting of cartilaginous and non-cartilaginous layers, and the former contains intact hypertrophic chondrocytes in uneroded lacunae. The purpose of this study is to determine the phenotype changes of uneroded hypertrophic chondrocytes. DESIGN: Immunohistochemical and ultrastructural examinations of the pericellular matrix of hypertrophic chondrocytes in the upper, middle, and lower regions of the mandibular condyle were conducted in aged c-src-deficient mice, using several antibodies of cartilage/bone marker proteins. RESULTS: Co-localisation of aggrecan, type I collagen, and dentin matrix protein-1 (DMP-1) or matrix extracellular phosphoprotein (MEPE) was detected in the pericellular matrix of the middle region. Ultrastructurally, granular substances in the pericellular matrix of the middle region were the remains of upper region chondrocytes, which were mixed with thick collagen fibrils. In the lower region, the width of the pericellular matrix and the amount of collagen fibrils were increased. Versican, type I collagen, DMP-1, and MEPE were detected in the osteocyte lacunae. Additionally, DMP-1 and MEPE were detected in the pericellular matrix of uneroded hypertrophic chondrocytes located in the lower, peripheral region of the mandibular condyle in younger c-src-deficient mice, but not in the aged wild-type mice. CONCLUSIONS: These results indicate that long-term survived, uneroded hypertrophic chondrocytes, at least in a part, acquire osteocytic characteristics.

Regulation of glycogen metabolism in gills and liver of the euryhaline tilapia (Oreochromis mossambicus) during acclimation to seawater.

Glucose, which plays a central role in providing energy for metabolism, is primarily stored as glycogen. The synthesis and degradation of glycogen are mainly initialized by glycogen synthase (GS) and glycogen phosphorylase (GP), respectively. The present study aimed to examine the glycogen metabolism in fish liver and gills during acute exposure to seawater. In tilapia (Oreochromis mossambicus) gill, GP, GS and glycogen were immunocytochemically colocalized in a specific group of glycogen-rich (GR) cells, which are adjacent to the gill's main ionocytes, mitochondrion-rich (MR) cells. Na+/K+-ATPase activity in the gills, protein expression and/or activity of GP and GS and the glycogen content of the gills and liver were examined in tilapia after their acute transfer from freshwater (FW) to 25 per thousand seawater (SW). Gill Na+/K+-ATPase activity rapidly increased immediately after SW transfer. Glycogen content in both the gills and liver were significantly depleted after SW transfer, but the depletion occurred earlier in gills than in the liver. Gill GP activity and protein expression were upregulated 1-3 h post-transfer and eventually recovered to the normal level as determined in the control group. At the same time, GS protein expression was downregulated. Similar changes in liver GP and GS protein expression were also observed but they occurred later at 6-12 h post-transfer. In conclusion, GR cells are initially stimulated to provide prompt energy for neighboring MR cells that trigger ion-secretion mechanisms. Several hours later, the liver begins to degrade its glycogen stores for the subsequent energy supply.

Diverse effects of c-src deficiency on molar tooth development and eruption in mice.

C-src deficiency is characterized by osteopetrosis due to impaired bone resorption by hypofunctional osteoclasts and the resultant failure of tooth eruption. In preliminary observations, we frequently encountered erupted molars in c-src deficient mice unlike in other osteopetrotic animals. Here we examine the effects of c-src deficiency on the development of molar teeth with an emphasis on the spatial relation of growing teeth with the surrounding bones. In c-src deficient mice, the magnitude of tooth impaction differed considerably among the types of molars; all maxillary 1st molars were totally impacted deep in the alveolar sockets, whereas most mandibular 1st molars fully erupted into oral cavity. Distribution of osteoclasts in the alveolar bone was identical among all types of molars, and electron microscopy revealed signs of bone resorbing activity in these osteoclasts despite the absence of a ruffled border. From early development, the alveolar space was much narrower in the upper molar tooth germs than in the lower ones in both wild type and homozygous animals, and particularly so in the upper 1st molars. Current observations thus indicate a significant contribution of "hypofunctional osteoclasts" in c-src deficient mice in molar tooth development except for the upper 1st molars, which appear to require highly functional osteoclasts to gain sufficient space for them to grow normally. Taken together, these findings on the seemingly tooth-type specific effects of c-src deficiency on the development and eruption of molar teeth in c-src deficient mice can be attributed to the given differential spatial relation of the respective tooth germs with the surrounding bones in the presence of hypofunctional osteoclasts.

Glycogen phosphorylase in glycogen-rich cells is involved in the energy supply for ion regulation in fish gill epithelia.

The molecular and cellular mechanisms behind glycogen metabolism and the energy metabolite translocation between mammal neurons and astrocytes have been well studied. A similar mechanism is proposed for rapid mobilization of local energy stores to support energy-dependent transepithelial ion transport in gills of the Mozambique tilapia (Oreochromis mossambicus). A novel gill glycogen phosphorylase isoform (tGPGG), which catalyzes the initial degradation of glycogen, was identified in branchial epithelial cells of O. mossambicus. Double in situ hybridization and immunocytochemistry demonstrated that tGPGG mRNA and glycogen were colocalized in glycogen-rich cells (GRCs), which surround ionocytes (labeled with a Na(+)-K(+)-ATPase antiserum) in gill epithelia. Concanavalin-A (a marker for the apical membrane) labeling indicated that GRCs and mitochondria-rich cells share the same apical opening. Quantitative real-time PCR analyses showed that tGPGG mRNA expression levels specifically responded to environmental salinity changes. Indeed, the glycogen content, glycogen phosphorylase (GP) protein level and total activity, and the density of tGPGG-expressing cells (i.e., GRCs) in fish acclimated to seawater (SW) were significantly higher than those in freshwater controls. Short-term acclimation to SW caused an evident depletion in the glycogen content of GRCs. Taken altogether, tGPGG expression in GRCs is stimulated by hyperosmotic challenge, and this may catalyze initial glycogen degradation to provide the adjacent ionocytes with energy to carry out iono- and osmoregulatory functions.

Formation of acellular cementum-like layers, with and without extrinsic fiber insertion, along inert bone surfaces of aging c-Src gene knockout mice.

To investigate the long-term effects of c-src deficiency on skeletal and dental tissues, we examined the lower jaws and long bones of c-src gene knockout (c-src KO) mice by histological and histochemical methods. Numerous multinucleated osteoclasts were distributed throughout the mandible in 5-wk-old c-src KO mice, but by 14 wk they had almost completely disappeared from the alveolar bone, leaving tartrate-resistant acid phosphatase (TRAP)-positive layers along the bone surface. Deposition of osteopontin-positive mineralized tissue, reminiscent of acellular afibrillar cementum (AAC), was confirmed along the TRAP-positive bone surface at 14 wk. The layer progressively thickened up to 21 months. A comparable mineralized layer was noted along the trabeculae of long bones as thickened cement lines. In the periostin-rich areas of jaw bones, but not in the long bones, portions of AAC-like mineralized layers were often replaced with and/or covered by acellular extrinsic fiber cementum (AEFC)-like tissue. These data suggest that the deposition of AAC-like mineralized tissue is a general phenomenon that may occur along inert or slowly remodeling bone surfaces under conditions characterized by reduced bone-resorbing activity, whereas the induction of AEFC-like tissue seems to be associated with the expression of certain molecules that are particularly abundant in the microenvironment of the periodontal ligament.

A novel domain in AMP-activated protein kinase causes glycogen storage bodies similar to those seen in hereditary cardiac arrhythmias.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that is activated by low cellular energy status and affects a switch away from energy-requiring processes and toward catabolism. While it is primarily regulated by AMP and ATP, high muscle glycogen has also been shown to repress its activation. Mutations in the gamma2 and gamma3 subunit isoforms lead to arrhythmias associated with abnormal glycogen storage in human heart and elevated glycogen in pig muscle, respectively. A putative glycogen binding domain (GBD) has now been identified in the beta subunits. Coexpression of truncated beta subunits lacking the GBD with alpha and gamma subunits yielded complexes that were active and normally regulated. However, coexpression of alpha and gamma with full-length beta caused accumulation of AMPK in large cytoplasmic inclusions that could be counterstained with anti-glycogen or anti-glycogen synthase antibodies. These inclusions were not affected by mutations that increased or abolished the kinase activity and were not observed by using truncated beta subunits lacking the GBD. Our results suggest that the GBD binds glycogen and can lead to abnormal glycogen-containing inclusions when the kinase is overexpressed. These may be related to the abnormal glycogen storage bodies seen in heart disease patients with gamma2 mutations.