Glycogen accumulation modulates life span in a mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the progressive loss of motor neurons in the spinal cord. Glial cells, including astrocytes and microglia, have been shown to contribute to neurodegeneration in ALS, and metabolic dysfunction plays an important role in the progression of the disease. Glycogen is a soluble polymer of glucose found at low levels in the central nervous system that plays an important role in memory formation, synaptic plasticity, and the prevention of seizures. However, its accumulation in astrocytes and/or neurons is associated with pathological conditions and aging. Importantly, glycogen accumulation has been reported in the spinal cord of human ALS patients and mouse models. In the present work, using the SOD1G93A mouse model of ALS, we show that glycogen accumulates in the spinal cord and brainstem during symptomatic and end stages of the disease and that the accumulated glycogen is associated with reactive astrocytes. To study the contribution of glycogen to ALS progression, we generated SOD1G93A mice with reduced glycogen synthesis (SOD1G93A GShet mice). SOD1G93A GShet mice had a significantly longer life span than SOD1G93A mice and showed lower levels of the astrocytic pro-inflammatory cytokine Cxcl10, suggesting that the accumulation of glycogen is associated with an inflammatory response. Supporting this, inducing an increase in glycogen synthesis reduced life span in SOD1G93A mice. Altogether, these results suggest that glycogen in reactive astrocytes contributes to neurotoxicity and disease progression in ALS.

Mutant IDH regulates glycogen metabolism from early cartilage development to malignant chondrosarcoma formation.

Chondrosarcomas are the most common malignancy of cartilage and are associated with somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 genes. Somatic IDH mutations are also found in its benign precursor lesion, enchondromas, suggesting that IDH mutations are early events in malignant transformation. Human mutant IDH chondrosarcomas and mutant Idh mice that develop enchondromas investigated in our studies display glycogen deposition exclusively in mutant cells from IDH mutant chondrosarcomas and Idh1 mutant murine growth plates. Pharmacologic blockade of glycogen utilization induces changes in tumor cell behavior, downstream energetic pathways, and tumor burden in vitro and in vivo. Mutant IDH1 interacts with hypoxia-inducible factor 1α (HIF1α) to regulate expression of key enzymes in glycogen metabolism. Here, we show a critical role for glycogen in enchondromas and chondrosarcomas, which is likely mediated through an interaction with mutant IDH1 and HIF1α.

Active Glycogen Synthase in the Liver Prevents High-Fat Diet-Induced Glucose Intolerance, Decreases Food Intake, and Lowers Body Weight.

Many lines of evidence demonstrate a correlation between liver glycogen content and food intake. We previously demonstrated that mice overexpressing protein targeting to glycogen (PTG) specifically in the liver-which have increased glycogen content in this organ-are protected from high-fat diet (HFD)-induced obesity by reduced food intake. However, the use of PTG to increase liver glycogen implies certain limitations. PTG stimulates glycogen synthesis but also inhibits the enzyme responsible for glycogen degradation. Furthermore, as PTG is a regulatory subunit of protein phosphatase 1 (PP1), which regulates many cellular functions, its overexpression could have side effects beyond the regulation of glycogen metabolism. Therefore, it is necessary to determine whether the direct activation of glycogen synthesis, without affecting its degradation or other cellular functions, has the same effects. To this end, we generated mice overexpressing a non-inactivatable form of glycogen synthase (GS) specifically in the liver (9A-MGSAlb mice). Control and 9a-MGSAlb mice were fed a standard diet (SD) or HFD for 16 weeks. Glucose tolerance and feeding behavior were analyzed. 9A-MGSAlb mice showed an increase in hepatic glycogen in fed and fasting conditions. When fed an HFD, these animals preserved their hepatic energy state, had a reduced food intake, and presented a lower body weight and fat mass than control animals, without changes in energy expenditure. Furthermore, 9A-MGSAlb animals showed improved glucose tolerance when fed an SD or HFD. Moreover, liver triacylglycerol levels that were increased after HFD feeding were lower in these mice. These results confirm that increased liver glycogen stores contribute to decreased appetite and improve glucose tolerance in mice fed an HFD. On the basis of our findings, strategies to preserve hepatic glycogen stores emerge as potential treatments for obesity and hyperglycemia.

Hepatic overexpression of protein targeting to glycogen attenuates obesity and improves hyperglycemia in db/db mice.

Increased liver glycogen content has been shown to reduce food intake, attenuate obesity, and improve glucose tolerance in a mouse model of high-fat diet (HFD)-induced obesity. Here we studied the contribution of liver glycogen to the regulation of obesity and glucose metabolism in a model of type 2 diabetes and obesity, namely the db/db mouse. To this end, we crossed db/db mice with animals overexpressing protein targeting to glycogen (PTG) in the liver to generate db/db mice with increased liver glycogen content (db/db-PTG). Hepatic PTG overexpression reduced food intake and fat weight and attenuated obesity and hyperglycemia in db/db mice. Db/db-PTG mice showed similar energy expenditure and physical activity to db/db mice. PTG overexpression reduced liver phosphoenolpyruvate carboxykinase (PEPCK) protein levels and repressed hepatic glucose production in db/db mice. Moreover, increased liver glycogen elevated hepatic ATP content in these animals. However, lipid metabolism was not modified by PTG overexpression. In conclusion, increased liver glycogen content ameliorates the diabetic and obesity phenotype in db/db mice.

Malin restoration as proof of concept for gene therapy for Lafora disease.

Lafora disease is a fatal neurodegenerative childhood dementia caused by loss-of-function mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of abnormal glycogen aggregates known as Lafora bodies (LBs) in the brain and other tissues. These aggregates are responsible for the pathological features of the disease. As a monogenic disorder, Lafora disease is a good candidate for gene therapy-based approaches. However, most patients are diagnosed after the appearance of the first symptoms and thus when LBs are already present in the brain. In this context, it was not clear whether the restoration of a normal copy of the defective gene (either laforin or malin) would prove effective. Here we evaluated the effect of restoring malin in a malin-deficient mouse model of Lafora disease as a proof of concept for gene replacement therapy. To this end, we generated a malin-deficient mouse in which malin expression can be induced at a certain time. Our results reveal that malin restoration at an advanced stage of the disease arrests the accumulation of LBs in brain and muscle, induces the degradation of laforin and glycogen synthase bound to the aggregates, and ameliorates neuroinflammation. These results identify malin restoration as the first therapeutic strategy to show effectiveness when applied at advanced stages of Lafora disease.

Lack of p62 Impairs Glycogen Aggregation and Exacerbates Pathology in a Mouse Model of Myoclonic Epilepsy of Lafora.

Lafora disease (LD) is a fatal childhood-onset dementia characterized by the extensive accumulation of glycogen aggregates-the so-called Lafora Bodies (LBs)-in several organs. The accumulation of LBs in the brain underlies the neurological phenotype of the disease. LBs are composed of abnormal glycogen and various associated proteins, including p62, an autophagy adaptor that participates in the aggregation and clearance of misfolded proteins. To study the role of p62 in the formation of LBs and its participation in the pathology of LD, we generated a mouse model of the disease (malinKO) lacking p62. Deletion of p62 prevented LB accumulation in skeletal muscle and cardiac tissue. In the brain, the absence of p62 altered LB morphology and increased susceptibility to epilepsy. These results demonstrate that p62 participates in the formation of LBs and suggest that the sequestration of abnormal glycogen into LBs is a protective mechanism through which it reduces the deleterious consequences of its accumulation in the brain.

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1.

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

Increased liver glycogen levels enhance exercise capacity in mice.

Muscle glycogen depletion has been proposed as one of the main causes of fatigue during exercise. However, few studies have addressed the contribution of liver glycogen to exercise performance. Using a low-intensity running protocol, here, we analyzed exercise capacity in mice overexpressing protein targeting to glycogen (PTG) specifically in the liver (PTGOE mice), which show a high concentration of glycogen in this organ. PTGOE mice showed improved exercise capacity, as determined by the distance covered and time ran in an extenuating endurance exercise, compared with control mice. Moreover, fasting decreased exercise capacity in control mice but not in PTGOE mice. After exercise, liver glycogen stores were totally depleted in control mice, but PTGOE mice maintained significant glycogen levels even in fasting conditions. In addition, PTGOE mice displayed an increased hepatic energy state after exercise compared with control mice. Exercise caused a reduction in the blood glucose concentration in control mice that was less pronounced in PTGOE mice. No changes were found in the levels of blood lactate, plasma free fatty acids, or ß-hydroxybutyrate. Plasma glucagon was elevated after exercise in control mice, but not in PTGOE mice. Exercise-induced changes in skeletal muscle were similar in both genotypes. These results identify hepatic glycogen as a key regulator of endurance capacity in mice, an effect that may be exerted through the maintenance of blood glucose levels.

Alteration of mitochondrial function in the livers of mice with glycogen branching enzyme deficiency.

Glycogen storage disease type IV (GSD IV) is caused by mutations in the glycogen branching enzyme gene (GBE1) that lead to the accumulation of aberrant glycogen in affected tissues, mostly in the liver. To determine whether dysfunctional glycogen metabolism in GSD IV affects other components of cellular bioenergetics, we studied mitochondrial function in heterozygous Gbe1 knockout (Gbe1+/-) mice. Mitochondria isolated from the livers of Gbe1+/- mice showed elevated respiratory complex I activity and increased reactive oxygen species production, particularly by respiratory chain complex III. These observations indicate that GBE1 deficiency leads to broader rearrangements in energy metabolism and that the mechanisms underlying GSD IV pathogenesis may include more than merely mechanical cell damage caused by the presence of glycogen aggregates.

Astrocytic glycogen accumulation drives the pathophysiology of neurodegeneration in Lafora disease.

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malinKO). Strikingly, blocking glycogen synthesis in astrocytes-thus impeding Lafora bodies accumulation in this cell type-prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malinKO model. Conversely, mice that over-accumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neurons.

Increasing hepatic glycogen moderates the diabetic phenotype in insulin-deficient Akita mice.

Hepatic glycogen metabolism is impaired in diabetes. We previously demonstrated that strategies to increase liver glycogen content in a high-fat-diet mouse model of obesity and insulin resistance led to a reduction in food intake and ameliorated obesity and glucose tolerance. These effects were accompanied by a decrease in insulin levels, but whether this decrease contributed to the phenotype observed in this animal was unclear. Here we sought to evaluate this aspect directly, by examining the long-term effects of increasing liver glycogen in an animal model of insulin-deficient and monogenic diabetes, namely the Akita mouse, which is characterized by reduced insulin production. We crossed Akita mice with animals overexpressing protein targeting to glycogen (PTG) in the liver to generate Akita mice with increased liver glycogen content (Akita-PTGOE). Akita-PTGOE animals showed lower glycemia, lower food intake, and decreased water consumption and urine output compared with Akita mice. Furthermore, Akita-PTGOE mice showed a restoration of the hepatic energy state and a normalization of gluconeogenesis and glycolysis back to nondiabetic levels. Moreover, hepatic lipogenesis, which is reduced in Akita mice, was reverted in Akita-PTGOE animals. These results demonstrate that strategies to increase liver glycogen content lead to the long-term reduction of the diabetic phenotype, independently of circulating insulin.

Neutrophils Fuel Effective Immune Responses through Gluconeogenesis and Glycogenesis.

Neutrophils can function and survive in injured and infected tissues, where oxygen and metabolic substrates are limited. Using radioactive flux assays and LC-MS tracing with U-13C glucose, glutamine, and pyruvate, we observe that neutrophils require the generation of intracellular glycogen stores by gluconeogenesis and glycogenesis for effective survival and bacterial killing. These metabolic adaptations are dynamic, with net increases in glycogen stores observed following LPS challenge or altitude-induced hypoxia. Neutrophils from patients with chronic obstructive pulmonary disease have reduced glycogen cycling, resulting in impaired function. Metabolic specialization of neutrophils may therefore underpin disease pathology and allow selective therapeutic targeting.

Suppression of glycogen synthesis as a treatment for Lafora disease: Establishing the window of opportunity.

Lafora disease (LD) is a fatal adolescence-onset neurodegenerative condition. The hallmark of LD is the accumulation of aberrant glycogen aggregates called Lafora bodies (LBs) in the brain and other tissues. Impeding glycogen synthesis from early embryonic stages by genetic suppression of glycogen synthase (MGS) in an animal model of LD prevents LB formation and ultimately the pathological manifestations of LD thereby indicating that LBs are responsible for the pathophysiology of the disease. However, it is not clear whether eliminating glycogen synthesis in an adult animal after LBs have already formed would halt or reverse the progression of LD. Herein we generated a mouse model of LD with inducible MGS suppression. We evaluated the effect of MGS suppression at different time points on LB accumulation as well as on the appearance of neuroinflammation, a pathologic trait of LD models. In the skeletal muscle, MGS suppression in adult LD mice blocked the formation of new LBs and reduced the number of glycogen aggregates. In the brain, early but not late MGS suppression halted the accumulation of LBs. However, the neuroinflammatory response was still present, as shown by the levels of reactive astrocytes, microglia and inflammatory cytokines. Our results confirm that MGS as a promising therapeutic target for LD and highlight the importance of an early diagnosis for effective treatment of the disease.

Pulmonary glycogen deficiency as a new potential cause of respiratory distress syndrome.

The glycogenin knockout mouse is a model of Glycogen Storage Disease type XV. These animals show high perinatal mortality (90%) due to respiratory failure. The lungs of glycogenin-deficient embryos and P0 mice have a lower glycogen content than that of wild-type counterparts. Embryonic lungs were found to have decreased levels of mature surfactant proteins SP-B and SP-C, together with incomplete processing of precursors. Furthermore, non-surviving pups showed collapsed sacculi, which may be linked to a significantly reduced amount of surfactant proteins. A similar pattern was observed in glycogen synthase1-deficient mice, which are devoid of glycogen in the lungs and are also affected by high perinatal mortality due to atelectasis. These results indicate that glycogen availability is a key factor for the burst of surfactant production required to ensure correct lung expansion at the establishment of air breathing. Our findings confirm that glycogen deficiency in lungs can cause respiratory distress syndrome and suggest that mutations in glycogenin and glycogen synthase 1 genes may underlie cases of idiopathic neonatal death.

Hallmarks of oxidative stress in the livers of aged mice with mild glycogen branching enzyme deficiency.

Glycogen branching enzyme (GBE1) introduces branching points in the glycogen molecule during its synthesis. Pathogenic GBE1 gene mutations lead to glycogen storage disease type IV (GSD IV), which is characterized by excessive intracellular accumulation of abnormal, poorly branched glycogen in affected tissues and organs, mostly in the liver. Using heterozygous Gbe1 knock-out mice (Gbe1+/-), we analyzed the effects of moderate GBE1 deficiency on oxidative stress in the liver. The livers of aged Gbe1+/- mice (22 months old) had decreased GBE1 protein levels, which caused a mild decrease in the degree of glycogen branching, but did not affect the tissue glycogen content. GBE1 deficiency was accompanied by increased protein carbonylation and elevated oxidation of the glutathione pool, indicating the existence of oxidative stress. Furthermore, we have observed increased levels of glutathione peroxidase and decreased activity of respiratory complex I in Gbe1+/- livers. Our data indicate that even mild changes in the degree of glycogen branching, which did not lead to excessive glycogen accumulation, may have broader effects on cellular bioenergetics and redox homeostasis. In young animals cellular homeostatic mechanisms are able to counteract those changes, while in aged tissues the changes may lead to increased oxidative stress.

Lack of Astrocytic Glycogen Alters Synaptic Plasticity but Not Seizure Susceptibility.

Brain glycogen is mainly stored in astrocytes. However, recent studies both in vitro and in vivo indicate that glycogen also plays important roles in neurons. By conditional deletion of glycogen synthase (GYS1), we previously developed a mouse model entirely devoid of glycogen in the central nervous system (GYS1Nestin-KO). These mice displayed altered electrophysiological properties in the hippocampus and increased susceptibility to kainate-induced seizures. To understand which of these functions are related to astrocytic glycogen, in the present study, we generated a mouse model in which glycogen synthesis is eliminated specifically in astrocytes (GYS1Gfap-KO). Electrophysiological recordings of awake behaving mice revealed alterations in input/output curves and impaired long-term potentiation, similar, but to a lesser extent, to those obtained with GYS1Nestin-KO mice. Surprisingly, GYS1Gfap-KO mice displayed no change in susceptibility to kainate-induced seizures as determined by fEPSP recordings and video monitoring. These results confirm the importance of astrocytic glycogen in synaptic plasticity.

Arousal-induced cortical activity triggers lactate release from astrocytes.

It has been suggested that, in states of arousal, release of noradrenaline and ß-adrenergic signalling affect long-term memory formation by stimulating astrocytic lactate production from glycogen. However, the temporal relationship between cortical activity and cellular lactate fluctuations upon changes in arousal remains to be fully established. Also, the role of ß-adrenergic signalling and brain glycogen metabolism on neural lactate dynamics in vivo is still unknown. Here, we show that an arousal-induced increase in cortical activity triggers lactate release into the extracellular space, and this correlates with a fast and prominent lactate dip in astrocytes. The immediate drop in astrocytic lactate concentration and the parallel increase in extracellular lactate levels underline an activity-dependent lactate release from astrocytes. Moreover, when ß-adrenergic signalling is blocked or the brain is depleted of glycogen, the arousal-evoked cellular lactate surges are significantly reduced. We provide in vivo evidence that cortical activation upon arousal triggers lactate release from astrocytes, a rise in intracellular lactate levels mediated by ß-adrenergic signalling and the mobilization of lactate from glycogen stores.

Scientists in education: A biomedical research institute's perspective.

The Institute for Research in Biomedicine (IRB Barcelona) is a world-class research center devoted to understanding fundamental questions about human health and disease. In addition to conducting multidisciplinary research of excellence, IRB Barcelona is committed to maintaining an open dialogue with the public about its work, fostering scientific vocations and promoting scientific culture in society. In this regard, the institute has several public engagement and science education initiatives to reach these goals. In this article, we highlight two projects directed toward primary school and secondary school students, respectively: the Tandem Project and the Crazy About Biomedicine Programme. Although each of these activities has slightly different objectives and target audiences, they share the commitment of the institute to contribute to the science education of young students. We believe that it is the responsibility of research institutes to promote these kinds of initiatives, which have the potential to spark passion for science and thus foster scientific vocations. The new generation of scientists is in our hands.

Maintenance of liver glycogen during long-term fasting preserves energy state in mice.

Glycogen shortage during fasting coincides with dramatic changes in hepatic adenine nucleotide levels. The aim of this work was to study the relevance of liver glycogen in the regulation of the hepatic energy state during food deprivation. To this end, we examined the response of mice with sustained increased liver glycogen content to prolonged fasting. In order to increase hepatic glycogen content, we generated mice that overexpress protein targeting to glycogen (PTG) in the liver (PTGOE  mice). Control and PTGOE  mice were fed ad libitum or fasted for 36 h. Upon fasting, PTGOE  mice retained significant hepatic glycogen stores and maintained hepatic energy status. Furthermore, we show that liver glycogen controls insulin sensitivity, gluconeogenesis, lipid metabolism, and ketogenesis upon nutrient deprivation.