[Show abstract][Hide abstract] ABSTRACT: Glycogen is present in the brain, where it has been found mainly in glial cells but not in neurons. Therefore, all physiologic roles of brain glycogen have been attributed exclusively to astrocytic glycogen. Working with primary cultured neurons, as well as with genetically modified mice and flies, here we report that-against general belief-neurons contain a low but measurable amount of glycogen. Moreover, we also show that these cells express the brain isoform of glycogen phosphorylase, allowing glycogen to be fully metabolized. Most importantly, we show an active neuronal glycogen metabolism that protects cultured neurons from hypoxia-induced death and flies from hypoxia-induced stupor. Our findings change the current view of the role of glycogen in the brain and reveal that endogenous neuronal glycogen metabolism participates in the neuronal tolerance to hypoxic stress.Journal of Cerebral Blood Flow & Metabolism advance online publication, 26 February 2014; doi:10.1038/jcbfm.2014.33.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 02/2014; · 5.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Liver and muscle glycogen content is reduced in diabetic patients but there is no information on the effect of diabetes on the glycogen content in the retinal pigment epithelium (RPE). The main aim of the study was to compare the glycogen content in the RPE between diabetic and non-diabetic human donors. Glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, as well as their isoforms, were also assessed. For this purpose, 44 human postmortem eye cups were included (22 from 11 type 2 diabetic and 22 from 11 non-diabetic donors matched by age). Human RPE cells cultured in normoglycemic and hyperglycemic conditions were also analyzed. Glycogen content as well as the mRNA, protein content and enzyme activity of GS and GP were determined. In addition, GS and GP isoforms were characterized. In the RPE from diabetic donors, as well as in RPE cells grown in hyperglycemic conditions, the glycogen content was increased. The increase in glycogen content was associated with an increase in GS without changes in GP levels. In RPE form human donors, the muscle GS isoform but not the liver GS isoform was detected. Regarding GP, the muscle and brain isoform of GP but not the liver GP isoform were detected. We conclude that glycogen storage is increased in the RPE of diabetic patients, and it is associated with an increase in GS activity. Further studies aimed at determining the role of glycogen deposits in the pathogenesis of diabetic retinopathy are warranted.
[Show abstract][Hide abstract] ABSTRACT: Lafora disease is a fatal neurodegenerative condition characterized by the accumulation of abnormal glycogen inclusions known as Lafora bodies. It is an autosomal recessive disorder caused by mutations in either the laforin or malin gene. To study whether glycogen is primarily responsible for the neurodegeneration in Lafora disease, we generated malin knockout mice with impaired (totally or partially) glycogen synthesis. These animals did not show the increase in markers of neurodegeneration, the impairments in electrophysiological properties of hippocampal synapses, nor the susceptibility to kainate-induced epilepsy seen in the malin knockout model. Interestingly, the autophagy impairment that has been described in malin knockout animals was also rescued in this double knockout model. Conversely, two other mouse models in which glycogen is over-accumulated in the brain independently of the lack of malin showed impairment in autophagy. Our findings reveal that glycogen accumulation accounts for the neurodegeneration and functional consequences seen in the malin knockout model, as well as the impaired autophagy. These results identify the regulation of glycogen synthesis as a key target for the treatment of Lafora disease.
Human Molecular Genetics 01/2014; · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pancreatic β-cells play a central role in type 2 diabetes (T2D) development, which is characterized by the progressive decline of the functional β-cell mass mainly associated with increased β-cell apoptosis. Thus, understanding how to enhance survival of β-cells is key for the management of T2D. The Insulin Receptor Substrate 2 (IRS2) protein is pivotal in mediating the Insulin/IGF signaling pathway in β-cells. In fact, IRS2 is critically required for β-cell compensation in conditions of increased insulin demand and for β-cell survival. Tungstate is a powerful anti-diabetic agent, which has been shown to promote β-cell recovery in toxin-induced diabetic rodent models. In this study, we investigated if tungstate could prevent the onset of diabetes in a scenario of dysregulated Insulin/IGF signaling and massive β-cell death. To this end, we treated mice deficient in IRS2 (Irs2(-/-)), which exhibit severe β-cell loss, with tungstate for 3 weeks. Tungstate normalized glucose tolerance in Irs2(-/-) mice, in correlation with increased β-cell mass, increased β-cell replication and a striking 3-fold reduction in β-cell apoptosis. Islets from treated Irs2-/- exhibited increased phosphorylated Erk1/2. Interestingly, tungstate repressed apoptosis-related genes in Irs2(-/-) islets in vitro and Erk1/2 blockade abolished some of these effects. Gene expression profiling evidenced a broad impact of tungstate on cell death pathways in islets from Irs2(-/-) mice, consistent with reduced apoptotic rates. Our results support that β-cell death can be arrested in the absence of IRS2 and that therapies aimed at reversing β-cell mass decline are potential strategies to prevent the progression to T2D.
AJP Endocrinology and Metabolism 11/2013; · 4.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through GS kinase-3 and glycogen-associated form of protein phosphatase 1. Here we initially performed mutagenesis analysis and identified a key residue (Arg582) required for activation of GYS2 by G6P. We then employed GYS2 Arg582Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation has been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A-mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P towards glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Both radiotherapy and most effective chemotherapeutic agents induce different types of DNA damage. Here we show that tungstate modulates cell response to DNA damaging agents. Cells treated with tungstate were more sensitive to etoposide, phleomycin and ionizing radiation (IR), all of which induce DNA double-strand breaks (DSBs). Tungstate also modulated the activation of the central DSB signalling kinase, ATM, in response to these agents. These effects required the functionality of the Mre11-Nbs1-Rad50 (MRN) complex and were mimicked by the inhibition of PP2A phosphatase. Therefore, tungstate may have adjuvant activity when combined with DNA-damaging agents in the treatment of several malignancies.
[Show abstract][Hide abstract] ABSTRACT: Glycogen is the only carbohydrate reserve of the brain, but its overall contribution to brain functions remains unclear. Although it has traditionally been considered as an emergency energetic reservoir, increasing evidence points to a role of glycogen in the normal activity of the brain. To address this long-standing question, we generated a brain-specific Glycogen Synthase knockout (GYS1(Nestin-KO)) mouse and studied the functional consequences of the lack of glycogen in the brain under alert behaving conditions. These animals showed a significant deficiency in the acquisition of an associative learning task and in the concomitant activity-dependent changes in hippocampal synaptic strength. Long-term potentiation (LTP) evoked in the hippocampal CA3-CA1 synapse was also decreased in behaving GYS1(Nestin-KO) mice. These results unequivocally show a key role of brain glycogen in the proper acquisition of new motor and cognitive abilities and in the underlying changes in synaptic strength.Journal of Cerebral Blood Flow & Metabolism advance online publication, 2 January 2013; doi:10.1038/jcbfm.2012.200.
Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 01/2013; · 5.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tungstate treatment ameliorates experimental diabetes by increasing liver glycogen deposition through an as yet unidentified mechanism. The signalling mechanism of tungstate was studied in CHOIR cells and primary cultured hepatocytes. This compound exerted its pro-glycogenic effects through a new G-protein-dependent and Tyr-Kinase Receptor-independent mechanism. Chemical or genetic disruption of G-protein signalling prevented the activation of the Ras/ERK cascade and the downstream induction of glycogen synthesis caused by tungstate. Thus, these findings unveil a novel non-canonical signalling pathway that leads to the activation of glycogen synthesis and that could be exploited as an approach to treat diabetes.
[Show abstract][Hide abstract] ABSTRACT: Under physiological conditions, most neurons keep glycogen synthase (GS) in an inactive form and do not show detectable levels of glycogen. Nevertheless, aberrant glycogen accumulation in neurons is a hallmark of patients suffering from Lafora disease or other polyglucosan disorders. Although these diseases are associated with mutations in genes involved in glycogen metabolism, the role of glycogen accumulation remains elusive. Here, we generated mouse and fly models expressing an active form of GS to force neuronal accumulation of glycogen. We present evidence that the progressive accumulation of glycogen in mouse and Drosophila neurons leads to neuronal loss, locomotion defects and reduced lifespan. Our results highlight glycogen accumulation in neurons as a direct cause of neurodegeneration.
EMBO Molecular Medicine 05/2012; 4(8):719-29. · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using a streptozotocin-induced type 1 diabetic rat model, we analyzed and separated the effects of hyperglycemia and hyperinsulinemia over the in vivo expression and subcellular localization of hepatic fructose 1,6-bisphosphatase (FBPase) in the multicellular context of the liver. Our data showed that FBPase subcellular localization was modulated by the nutritional state in normal but not in diabetic rats. By contrast, the liver zonation was not affected in any condition. In healthy starved rats, FBPase was localized in the cytoplasm of hepatocytes, whereas in healthy re-fed rats it was concentrated in the nucleus and the cell periphery. Interestingly, despite the hyperglycemia, FBPase was unable to accumulate in the nucleus in hepatocytes from streptozotocin-induced diabetic rats, suggesting that insulin is a critical in vivo modulator. This idea was confirmed by exogenous insulin supplementation to diabetic rats, where insulin was able to induce the rapid accumulation of FBPase within the hepatocyte nucleus. Besides, hepatic FBPase was found phosphorylated only in the cytoplasm, suggesting that the phosphorylation state is involved in the nuclear translocation. In conclusion, insulin and not hyperglycemia plays a crucial role in the nuclear accumulation of FBPase in vivo and may be an important regulatory mechanism that could account for the increased endogenous glucose production of liver of diabetic rodents.
Journal of Cellular Biochemistry 03/2012; 113(3):848-56. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tungstate counteracts diabetes and obesity in animal models, but its molecular mechanisms remain elusive. Our Saccharomyces cerevisiae-based approach has found that tungstate alleviated the growth defect induced by nutrient stress and enhanced the activation of the GCN pathway. Tungstate relieved the sensitivity to starvation of a gcn2-507 yeast hypomorphic mutant, indicating that tungstate modulated the GCN pathway downstream of Gcn2p. Interestingly, tungstate inhibited Glc7p and PP1 phosphatase activity, both negative regulators of the GCN pathway in yeast and humans, respectively. Accordingly, overexpression of a dominant-negative Glc7p mutant in yeast mimicked tungstate effects. Therefore tungstate alleviates nutrient stress in yeast by in vivo inhibition of Glc7p. These data uncover a potential role for tungstate in the treatment of PP1 and GCN related diseases.
[Show abstract][Hide abstract] ABSTRACT: Recombinant muscle GYS1 (glycogen synthase 1) and recombinant liver GYS2 were phosphorylated by recombinant AMPK (AMP-activated protein kinase) in a time-dependent manner and to a similar stoichiometry. The phosphorylation site in GYS2 was identified as Ser7, which lies in a favourable consensus for phosphorylation by AMPK. Phosphorylation of GYS1 or GYS2 by AMPK led to enzyme inactivation by decreasing the affinity for both UDP-Glc (UDP-glucose) [assayed in the absence of Glc-6-P (glucose-6-phosphate)] and Glc-6-P (assayed at low UDP-Glc concentrations). Incubation of freshly isolated rat hepatocytes with the pharmacological AMPK activators AICA riboside (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) or A769662 led to persistent GYS inactivation and Ser7 phosphorylation, whereas inactivation by glucagon treatment was transient. In hepatocytes from mice harbouring a liver-specific deletion of the AMPK catalytic α1/α2 subunits, GYS2 inactivation by AICA riboside and A769662 was blunted, whereas inactivation by glucagon was unaffected. The results suggest that GYS inactivation by AMPK activators in hepatocytes is due to GYS2 Ser7 phosphorylation.
[Show abstract][Hide abstract] ABSTRACT: Oral administration of sodium tungstate has shown hyperglycemia-reducing activity in several animal models of diabetes. We present new insights into the mechanism of action of tungstate.
We studied protein expression and phosphorylation in the liver of STZ rats, a type I diabetes model, treated with sodium tungstate in the drinking water (2 mg/ml) and in primary cultured-hepatocytes, through Western blot and Real Time PCR analysis.
Tungstate treatment reduces the expression of gluconeogenic enzymes (PEPCK, G6Pase, and FBPase) and also regulates transcription factors accountable for the control of hepatic metabolism (c-jun, c-fos and PGC1α). Moreover, ERK, p90rsk and GSK3, upstream kinases regulating the expression of c-jun and c-fos, are phosphorylated in response to tungstate. Interestingly, PKB/Akt phosphorylation is not altered by the treatment. Several of these observations were reproduced in isolated rat hepatocytes cultured in the absence of insulin, thereby indicating that those effects of tungstate are insulin-independent.
Here we show that treatment with tungstate restores the phosphorylation state of various signaling proteins and changes the expression pattern of metabolic enzymes.
PLoS ONE 01/2012; 7(8):e42305. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV(+)) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV(+) interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin-malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.
EMBO Molecular Medicine 08/2011; 3(11):667-81. · 7.80 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycogen deposition is impaired in diabetes, thus contributing to the development of hyperglycaemia. Several glucose-lowering strategies have attempted to increase liver glycogen deposition by modulating targets, which eventually trigger the activation of liver glycogen synthase (LGS). However, these targets also alter several other biological processes, and therefore their therapeutic use may be limited. Here we tested the approach of directly activating LGS and evaluated the potential of this strategy as a possible treatment for diabetes.
In this study, we examined the efficacy of directly overproducing a constitutively active form of LGS in the liver to ameliorate streptozotocin-induced diabetes in rats.
Activated mutant LGS overproduction in the liver of streptozotocin-induced diabetic rats normalised liver glycogen content, despite low levels of glucokinase and circulating insulin. Moreover, this overproduction led to a decrease in food intake and in the production of the main gluconeogenic enzymes, glucose-6-phosphatase, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase. The resulting combined effect was a reduction in hyperglycaemia.
The restoration of liver glycogen ameliorated diabetes and therefore is considered a potential strategy for the treatment of this disease.
[Show abstract][Hide abstract] ABSTRACT: It is known that oral administration of sodium tungstate preserves the pancreatic beta cell function in diabetic rats. Healthy and streptozotocin-induced diabetic rats were treated with sodium tungstate for one, three or six weeks, after which the species of W in serum, were analysed. An increase in serum W with treatment time was observed. After six weeks, the serum W concentration in diabetic rats (70 mg L(-1)) was about 4.6 times higher than in healthy specimens. This different behaviour was also observed for Cu accumulation, while the Zn pattern follows the contrary. The patterns observed in the retention of Cu and Zn may be attributable to a normalization of glycaemia. The speciation analysis of W was performed using 2D separations, including an immunoaffinity packing and a SEC (Size Exclusion Chromatography) column coupled to an ICP-MS (Inductively Coupled Plasma Mass Spectrometry) for elemental detection. Ultrafiltration data together with SEC-ICP-MS results proved that around 80% of serum W was bound to proteins, the diabetic rats registering a higher W content than their healthy counterparts. Most of the protein-bound W was due to a complex with albumin. An unknown protein with a molecular weight higher than 100 kDa was also found to bind a small amount of W (about 2%). MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) analysis of the desalted and concentrated chromatographic fractions confirmed albumin as the main protein bound to tungstate in rat serum, while no binding to transferrin (Tf) was detected. The interaction between glutathione and W was also evaluated using standard solutions; however, the formation of complexes was not observed. The stability of the complexes between W and proteins when subjected to more stringent procedures, like those used in proteomic methodologies (denaturing with urea or SDS, boiling, sonication, acid media, reduction with β-mercaptoethanol (BME) or DTT (dithiotreitol) and alkylation with iodoacetamide (IAA), was also evaluated. Our results indicate that the stability of the complexes between W and proteins is not too high enough to remain unaltered during protein separation by SDS-PAGE in denaturing and reducing conditions. However, the procedures for in-solution tryptic digestion and for ESI-MS analysis in MeOH/H(2)O/with 0.1% formic acid could be used for protein identification without large loss of binding between W and proteins.
[Show abstract][Hide abstract] ABSTRACT: The main aim of the present work was to test the effects of glucose and fructose on the phosphorylation levels of proteins linked to the control of overall sperm function in two species with very different metabolic characteristics, dog and boar. Incubation of dog spermatozoa with 10mM glucose increased serine phosphorylation of proteins related to cell cycle and signal transduction including cyclins B and E, Cdk2, Cdk6, Cdc6, PYK2, c-kit, Raf-1, TRK and several protein phosphatases. Incubation of dog spermatozoa with 10mM fructose decreased serine phosphorylation levels of cyclins B and D3, Cdk1/Cdc2, Cdk2, Cdk6, Akt, PI3 kinase, ERK-1 and protein kinase C. Incubation of boar spermatozoa with glucose or fructose did not modify any of the phosphorylation patterns studied. Given that one important difference between dog and boar spermatozoa is the presence of glucokinase (GK) in dog but not in boar, GK-transfected COS7 cells were incubated with either 10mM glucose or 10mM fructose. Incubation of GK-transfected cells with fructose decreased serine phosphorylation of cyclin A, ERK-2 and Hsp-70. In contrast, incubation of control COS7 cells with fructose increased serine phosphorylation of Cdk6, Cdk1/Cdc2, protein kinase C and Hsp-70. Incubation with glucose did not induce any significant effect. Our results indicate that monosaccharides act as signalling compounds in dog spermatozoa after ejaculation through changes in the phosphorylation levels of specific proteins. One of the factors that may be related to the action of sugars is the equilibrium of the total sperm hexokinase activity, in which the presence or absence of GK appears to be relevant.
Reproduction Fertility and Development 04/2011; 23(3):468-80. · 2.58 Impact Factor