Beyond being an energy supplier, ATP synthase is a sculptor of mitochondrial cristae.

Mitochondrial F1FO-ATP synthase plays a key role in cellular bioenergetics; this enzyme is present in all eukaryotic linages except in amitochondriate organisms. Despite its ancestral origin, traceable to the alpha proteobacterial endosymbiotic event, the actual structural diversity of these complexes, due to large differences in their polypeptide composition, reflects an important evolutionary divergence between eukaryotic lineages. We discuss the effect of these structural differences on the oligomerization of the complex and the shape of mitochondrial cristae.

Coffee Increases Post-Exercise Muscle Glycogen Recovery in Endurance Athletes: A Randomized Clinical Trial.

Coffee is one of the most widely consumed beverages worldwide and caffeine is known to improve performance in physical exercise. Some substances in coffee have a positive effect on glucose metabolism and are promising for post-exercise muscle glycogen recovery. We investigated the effect of a coffee beverage after exhaustive exercise on muscle glycogen resynthesis, glycogen synthase activity and glycemic and insulinemic response in a double-blind, crossover, randomized clinical trial. Fourteen endurance-trained men performed an exhaustive cycle ergometer exercise to deplete muscle glycogen. The following morning, participants completed a second cycling protocol followed by a 4-h recovery, during which they received either test beverage (coffee + milk) or control (milk) and a breakfast meal, with a simple randomization. Blood samples and muscle biopsies were collected at the beginning and by the end of recovery. Eleven participants were included in data analysis (age: 39.0 ± 6.0 years; BMI: 24.0 ± 2.3 kg/m2; VO2max: 59.9 ± 8.3 mL·kg-1·min-1; PPO: 346 ± 39 W). The consumption of coffee + milk resulted in greater muscle glycogen recovery (102.56 ± 18.75 vs. 40.54 ± 18.74 mmol·kg dw-1; p = 0.01; d = 0.94) and greater glucose (p = 0.02; d = 0.83) and insulin (p = 0.03; d = 0.76) total area under the curve compared with control. The addition of coffee to a beverage with adequate amounts of carbohydrates increased muscle glycogen resynthesis and the glycemic and insulinemic response during the 4-h recovery after exhaustive cycling exercise.

Identification of a novel starch synthase III from the picoalgae Ostreococcus tauri.

Hydrosoluble glycogen is the major energy storage compound in bacteria, archaea, fungi, and animal cells. In contrast, photosynthetic eukaryotes have evolved to build a highly organized semicrystalline granule of starch. Several enzymes are involved in polysaccharide synthesis, among which glycogen or starch synthase catalyze the elongation of the α-1,4-glucan chain. Ostreococcus tauri, accumulates a single starch granule and contains three starch synthase III (SSIII) isoforms, known as OsttaSSIII-A, OsttaSSIII-B and OsttaSSIII-C. After amino acids sequence analysis we found that OsttaSSIII-C lacks starch-binding domains, being 49% identical to the catalytic region of the SSIII from Arabidopsis thaliana and 32% identical to the entire Escherichia coli glycogen synthase. The recombinant, highly purified OsttaSSIII-C exhibited preference to use as a primer branched glycans (such as rabbit muscle glycogen and amylopectin), rather than amylose. Also, the enzyme displayed a high affinity toward ADP-glucose. We found a marked conservation of the amino acids located in the catalytic site, and specifically determined the role of residues R270, K275 and E352 by site-directed mutagenesis. Results show that these residues are important for OsttaSSIII-C activity, suggesting a strong similarity between the active site of the O. tauri SSIII-C isoform and other bacterial glycogen synthases.

Glycogen Synthase in Sertoli Cells: More Than Glycogenesis?

Sertoli cell metabolism actively maintains the nutritional needs of germ cells. It has been described that after glucose incorporation in Sertoli cells, less than 1% is converted to glycogen suggesting low levels of glycogen synthase activity. Phosphorylation of muscle glycogen synthase (MGS) at serine 640 (pS640MGS) decreases its activity, and this form of the enzyme was discovered as a non-ribosomal protein that modulates the translation of a subset of transcripts in HeLa cells. The aim of our study was to functionally characterize MGS in cultured Sertoli cells, as well as to explore this new feature related to RNA molecules. We detected MGS in the cytoplasm of Sertoli cells as well as in the nuclei. The activity rates of the enzyme were extremely low indicating that MGS is expressed but almost inactive. Protein targeting to glycogen (PTG) overexpression was performed to activate MGS by dephosphorylation. PTG induced glycogen synthesis massively, confirming that this enzyme is present but inactive. This finding correlates with high levels of pS640MGS, which were assayed by phosphatase treatment. To explore a putative new function for MGS in Sertoli cells, we performed RNA immunoprecipitation coupled to microarray studies. The results revealed that MGS co-immunoprecipitated with the several mRNAs and also rRNAs. These findings indicate that MGS is expressed Sertoli cells but in an inactive form, and also support a possibly novel feature of this metabolic enzyme associated with RNA-related molecules. J. Cell. Biochem. 117: 2597-2607, 2016. © 2016 Wiley Periodicals, Inc.

Lithium Induces Glycogen Accumulation in Salivary Glands of the Rat.

Lithium is administered for the treatment of mood and bipolar disorder. The aim of this study was to verify whether treatment with different concentrations of lithium may affect the glycogen metabolism in the salivary glands of the rats when compared with the liver. Mobilization of glycogen in salivary glands is important for the process of secretion. Two sets of experiments were carried out, that is, in the first, the rats received drinking water supplemented with LiCl (38,25 and 12 mM of LiCl for 15 days) and the second experiment was carried out by intraperitoneal injection of LiCl solution (12 mg/kg and 45 mg LiCl/kg body weight) for 3 days. The active form of glycogen phosphorylase was not affected by treatment with LiCl considering the two experiments. The active form of glycogen synthase presented higher activity in the submandibular glands of rats treated with 25 and 38 mM LiCl and in the liver, with 25 mM LiCl. Glycogen level was higher than that of control in the submandibular glands of rats receiving 38 and 12 mM LiCl, in the parotid of rats receiving 25 and 38 mM, and in the liver of rats receiving 12 mM LiCl. The absolute value of glycogen for the submandibular treated with 25 mM LiCl, and the liver treated with 38 mM LiCl, was higher than the control value, although not statistically significant for these tissues. No statistically significant difference was found in the submandibular and parotid salivary glands for protein concentration when comparing experimental and control groups. We concluded that LiCl administered to rats influences the metabolism of glycogen in salivary glands.

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator.

The transcription factor CreA/Mig1/CRE-1 is a repressor protein that regulates the use of alternative carbon sources via a mechanism known as Carbon Catabolite Repression (CCR). In Saccharomyces cerevisiae, Mig1 recruits the complex Ssn6-Tup1, the Neurospora crassa RCM-1 and RCO-1 orthologous proteins, respectively, to bind to promoters of glucose-repressible genes. We have been studying the regulation of glycogen metabolism in N. crassa and the identification of the RCO-1 corepressor as a regulator led us to investigate the regulatory role of CRE-1 in this process. Glycogen content is misregulated in the rco-1(KO), rcm-1(RIP) and cre-1(KO) strains, and the glycogen synthase phosphorylation is decreased in all strains, showing that CRE-1, RCO-1 and RCM-1 proteins are involved in glycogen accumulation and in the regulation of GSN activity by phosphorylation. We also confirmed the regulatory role of CRE-1 in CCR and its nuclear localization under repressing condition in N. crassa. The expression of all glycogenic genes is misregulated in the cre-1(KO) strain, suggesting that CRE-1 also controls glycogen metabolism by regulating gene expression. The existence of a high number of the Aspergillus nidulans CreA motif (5'-SYGGRG-3') in the glycogenic gene promoters led us to analyze the binding of CRE-1 to some DNA motifs both in vitro by DNA gel shift and in vivo by ChIP-qPCR analysis. CRE-1 bound in vivo to all motifs analyzed demonstrating that it down-regulates glycogen metabolism by controlling gene expression and GSN phosphorylation.

Allosteric regulation of the partitioning of glucose-1-phosphate between glycogen and trehalose biosynthesis in Mycobacterium tuberculosis.

BACKGROUND: Mycobacterium tuberculosis is a pathogenic prokaryote adapted to survive in hostile environments. In this organism and other Gram-positive actinobacteria, the metabolic pathways of glycogen and trehalose are interconnected. RESULTS: In this work we show the production, purification and characterization of recombinant enzymes involved in the partitioning of glucose-1-phosphate between glycogen and trehalose in M. tuberculosis H37Rv, namely: ADP-glucose pyrophosphorylase, glycogen synthase, UDP-glucose pyrophosphorylase and trehalose-6-phosphate synthase. The substrate specificity, kinetic parameters and allosteric regulation of each enzyme were determined. ADP-glucose pyrophosphorylase was highly specific for ADP-glucose while trehalose-6-phosphate synthase used not only ADP-glucose but also UDP-glucose, albeit to a lesser extent. ADP-glucose pyrophosphorylase was allosterically activated primarily by phosphoenolpyruvate and glucose-6-phosphate, while the activity of trehalose-6-phosphate synthase was increased up to 2-fold by fructose-6-phosphate. None of the other two enzymes tested exhibited allosteric regulation. CONCLUSIONS: Results give information about how the glucose-1-phosphate/ADP-glucose node is controlled after kinetic and regulatory properties of key enzymes for mycobacteria metabolism. GENERAL SIGNIFICANCE: This work increases our understanding of oligo and polysaccharides metabolism in M. tuberculosis and reinforces the importance of the interconnection between glycogen and trehalose biosynthesis in this human pathogen.

Over-expression of muscle glycogen synthase in human diabetic nephropathy.

Diabetic nephropathy (DN) is a major complication of diabetic patients and the leading cause of end-stage renal disease. Glomerular dysfunction plays a critical role in DN, but deterioration of renal function also correlates with tubular alterations. Human DN is characterized by glycogen accumulation in tubules. Although this pathological feature has long been recognized, little information exists about the triggering mechanism. In this study, we detected over-expression of muscle glycogen synthase (MGS) in diabetic human kidney. This enhanced expression suggests the participation of MGS in renal metabolic changes associated with diabetes. HK2 human renal cell line exhibited an intrinsic ability to synthesize glycogen, which was enhanced after over-expression of protein targeting to glycogen. A correlation between increased glycogen amount and cell death was observed. Based on a previous transcriptome study on human diabetic kidney disease, significant differences in the expression of genes involved in glycogen metabolism were analyzed. We propose that glucose, but not insulin, is the main modulator of MGS activity in HK2 cells, suggesting that blood glucose control is the best approach to modulate renal glycogen-induced damage during long-term diabetes.

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

Dietary Salba (Salvia hispanica L) seed rich in α-linolenic acid improves adipose tissue dysfunction and the altered skeletal muscle glucose and lipid metabolism in dyslipidemic insulin-resistant rats.

This work reports the effect of dietary Salba (chia) seed rich in n-3 α-linolenic acid on the morphological and metabolic aspects involved in adipose tissue dysfunction and the mechanisms underlying the impaired glucose and lipid metabolism in the skeletal muscle of rats fed a sucrose-rich diet (SRD). Rats were fed a SRD for 3 months. Thereafter, half the rats continued with SRD while in the other half, corn oil (CO) was replaced by chia seed for 3 months (SRD+chia). In control group, corn starch replaced sucrose. The replacement of CO by chia seed in the SRD reduced adipocyte hypertrophy, cell volume and size distribution, improved lipogenic enzyme activities, lipolysis and the anti-lipolytic action of insulin. In the skeletal muscle lipid storage, glucose phosphorylation and oxidation were normalized. Chia seed reversed the impaired insulin stimulated glycogen synthase activity, glycogen, glucose-6-phosphate and GLUT-4 protein levels as well as insulin resistance and dyslipidemia.

Putative role of glycogen as a peripheral biomarker of GSK3ß activity.

Glycogen synthase kinase 3-ß (GSK3ß) has a pivotal role in several intracellular signaling cascades that are involved in gene transcription, cytoskeletal reorganization, energy metabolism, cell cycle regulation, and apoptosis. This kinase has pleiotropic functions, and the importance of its activity has recently been shown in neurons and platelets. In addition to its regulatory function in several physiological events, changes in GSK3ß activity have been associated with many psychiatric and neurodegenerative illnesses, such as Alzheimer's disease, schizophrenia and autism-spectrum disorders. Beside the reports of its involvement in several pathologies, it has become increasingly apparent that GSK3ß might be a common therapeutic target for different classes of psychiatric drugs, and also that the GSK3ß ratio may be a useful parameter to determine the biochemical changes that might occur during antidepressant treatment. Although GSK3ß is commonly described as a key enzyme in a plethora of signaling cascades, originally it was identified as playing an important role in the regulation of glycogen synthesis, given its ability to inactivate glycogen synthase (GS) by phosphorylation. Acting as a constitutively active kinase, GSK3ß phosphorylates GS, which results in a decrease of glycogen production. GSK3ß phosphorylation increases glycogen synthesis and storage, while its dephosphorylation decreases glycogen synthesis. Inactivation of GSK3ß leads to dephosphorylation of GS and increase in glycogen synthesis in the adipose tissue, muscle and liver. Glycogen levels are reduced by antidepressant treatment, and this effect seems to be related to an effect of drugs on GSK3ß activity. Peripherally, glycogen is also abundantly found in platelets, where it is considered a major energy source, required for a variety of its functions, including the release reaction. Recently, analysis of platelets from patients with late-life major depression showed that active forms of GSK3ß expression were upregulated by continuous treatment with sertraline. Here, we hypothesized that the quantification of glycogen in platelets might be used as a peripheral biomarker of GSK3ß activity. Since it has been recently demonstrated that the modulation of GSK3ß activity causes changes in glycogen stores, the glycogen levels in platelets could be used to assay the effects of drugs that have this kinase as a target, or diseases where its activity is affected. In conclusion, we hypothesized that the determination of glycogen peripherally may be useful to indicate a change in the activity of this enzyme, providing a faster and non-invasive approach to guide the therapeutic procedures for the patient.

Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs).

Glycogen and starch, the major storage carbohydrate in most living organisms, result mainly from the action of starch or glycogen synthases (SS or GS, respectively, EC 2.4.1.21). SSIII from Arabidopsis thaliana is an SS isoform with a particular modular organization: the C-terminal highly conserved glycosyltransferase domain is preceded by a unique specific region (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of polysaccharide degrading enzymes. N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. On the other hand, GS from Agrobacterium tumefaciens has a simple primary structure organization, characterized only by the highly conserved glycosyltransferase domain and lacking SBDs. To further investigate the functional role of A. thaliana SSIII-SD, three chimeric proteins were constructed combining the SBDs from A. thaliana with the GS from A. tumefaciens. Recombinant proteins were expressed in and purified to homogeneity from Escherichia coli cells in order to be kinetically characterized. Furthermore, we tested the ability to restore in vivo glycogen biosynthesis in transformed E. coli glgA(-) cells, deficient in GS. Results show that the D3-GS chimeric enzyme showed increased capacity of glycogen synthesis in vivo with minor changes in its kinetics parameters compared to GS.

Immunohistochemical localization of glycogen synthase and GSK3ß: control of glycogen content in retina.

Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3ß (GSK3ß), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3ß in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3ß in the synaptic layers and photoreceptor cells suggests an important role of GSK3ß regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina.

Muscle glycogen synthase isoform is responsible for testicular glycogen synthesis: glycogen overproduction induces apoptosis in male germ cells.

Glycogen is the main source of glucose for many biological events. However, this molecule may have other functions, including those that have deleterious effects on cells. The rate-limiting enzyme in glycogen synthesis is glycogen synthase (GS). It is encoded by two genes, GYS1, expressed in muscle (muscle glycogen synthase, MGS) and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase, LGS). Expression of GS and its activity have been widely studied in many tissues. To date, it is not clear which GS isoform is responsible for glycogen synthesis and the role of glycogen in testis. Using RT-PCR, Western blot and immunofluorescence, we have detected expression of MGS but not LGS in mice testis during development. We have also evaluated GS activity and glycogen storage at different days after birth and we show that both GS activity and levels of glycogen are higher during the first days of development. Using RT-PCR, we have also shown that malin and laforin are expressed in testis, key enzymes for regulation of GS activity. These proteins form an active complex that regulates MGS by poly-ubiquitination in both Sertoli cell and male germ cell lines. In addition, PTG overexpression in male germ cell line triggered apoptosis by caspase3 activation, proposing a proapoptotic role of glycogen in testis. These findings suggest that GS activity and glycogen synthesis in testis could be regulated and a disruption of this process may be responsible for the apoptosis and degeneration of seminiferous tubules and possible cause of infertility.

Ambient pH controls glycogen levels by regulating glycogen synthase gene expression in Neurospora crassa. New insights into the pH signaling pathway.

Glycogen is a polysaccharide widely distributed in microorganisms and animal cells and its metabolism is under intricate regulation. Its accumulation in a specific situation results from the balance between glycogen synthase and glycogen phosphorylase activities that control synthesis and degradation, respectively. These enzymes are highly regulated at transcriptional and post-translational levels. The existence of a DNA motif for the Aspergillus nidulans pH responsive transcription factor PacC in the promoter of the gene encoding glycogen synthase (gsn) in Neurospora crassa prompted us to investigate whether this transcription factor regulates glycogen accumulation. Transcription factors such as PacC in A. nidulans and Rim101p in Saccharomyces cerevisiae play a role in the signaling pathway that mediates adaptation to ambient pH by inducing the expression of alkaline genes and repressing acidic genes. We showed here that at pH 7.8 pacC was over-expressed and gsn was down-regulated in wild-type N. crassa coinciding with low glycogen accumulation. In the pacC(KO) strain the glycogen levels and gsn expression at alkaline pH were, respectively, similar to and higher than the wild-type strain at normal pH (5.8). These results characterize gsn as an acidic gene and suggest a regulatory role for PACC in gsn expression. The truncated recombinant protein, containing the DNA-binding domain specifically bound to a gsn DNA fragment containing the PacC motif. DNA-protein complexes were observed with extracts from cells grown at normal and alkaline pH and confirmed by ChIP-PCR analysis. The PACC present in these extracts showed equal molecular mass, indicating that the protein is already processed at normal pH, in contrast to A. nidulans. Together, these results show that the pH signaling pathway controls glycogen accumulation by regulating gsn expression and suggest the existence of a different mechanism for PACC activation in N. crassa.

Control of glycogen content in retina: allosteric regulation of glycogen synthase.

Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity. Glycogen and G-6-P content were studied in ex vivo retinas from normal, fasted, streptozotocin-treated, and insulin-induced hypoglycemic rats. Expression levels of GS and its phosphorylated form were also analyzed. Ex vivo retina from normal rats showed low G-6-P (14±2 pmol/mg protein) and glycogen levels (43±3 nmol glycosyl residues/mg protein), which were increased 6 and 3 times, respectively, in streptozotocin diabetic rats. While no changes in phosphorylated GS levels were observed in any condition tested, a positive correlation was found between G-6-P levels with GS activity and glycogen content. The results indicated that in vivo, retina glycogen may act as an immediately accessible energy reserve and that its content was controlled primarily by G-6-P allosteric activation of GS. Therefore, under hypoglycemic situations retina energy supply is strongly compromised and could lead to the alterations observed in type 1 diabetes.

Characterization of recombinant UDP- and ADP-glucose pyrophosphorylases and glycogen synthase to elucidate glucose-1-phosphate partitioning into oligo- and polysaccharides in Streptomyces coelicolor.

Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V(max) in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V(max) of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.

An enzyme-coupled continuous spectrophotometric assay for glycogen synthases.

The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.

A genome-wide screen for Neurospora crassa transcription factors regulating glycogen metabolism.

Transcription factors play a key role in transcription regulation as they recognize and directly bind to defined sites in promoter regions of target genes, and thus modulate differential expression. The overall process is extremely dynamic, as they have to move through the nucleus and transiently bind to chromatin in order to regulate gene transcription. To identify transcription factors that affect glycogen accumulation in Neurospora crassa, we performed a systematic screen of a deletion strains set generated by the Neurospora Knockout Project and available at the Fungal Genetics Stock Center. In a wild-type strain of N. crassa, glycogen content reaches a maximal level at the end of the exponential growth phase, but upon heat stress the glycogen content rapidly drops. The gene encoding glycogen synthase (gsn) is transcriptionally down-regulated when the mycelium is exposed to the same stress condition. We identified 17 deleted strains having glycogen accumulation profiles different from that of the wild-type strain under both normal growth and heat stress conditions. Most of the transcription factors identified were annotated as hypothetical protein, however some of them, such as the PacC, XlnR, and NIT2 proteins, were biochemically well-characterized either in N. crassa or in other fungi. The identification of some of the transcription factors was coincident with the presence of DNA-binding motifs specific for the transcription factors in the gsn 5'-flanking region, and some of these DNA-binding motifs were demonstrated to be functional by Electrophoretic Mobility Shift Assay (EMSA) experiments. Strains knocked-out in these transcription factors presented impairment in the regulation of gsn expression, suggesting that the transcription factors regulate glycogen accumulation by directly regulating gsn gene expression. Five selected mutant strains showed defects in cell cycle progression, and two transcription factors were light-regulated. The results indicate that there are connections linking different cellular processes, such as metabolism control, biological clock, and cell cycle progression.

Control of carbohydrate metabolism in an anoxia-tolerant nervous system.

Anoxia-tolerant animal models are crucial to understand protective mechanisms during low oxygen excursions. As glycogen is the main fermentable fuel supporting energy production during oxygen tension reduction, understanding glycogen metabolism can provide important insights about processes involved in anoxia survival. In this report we studied carbohydrate metabolism regulation in the central nervous system (CNS) of an anoxia-tolerant land snail during experimental anoxia exposure and subsequent reoxygenation. Glucose uptake, glycogen synthesis from glucose, and the key enzymes of glycogen metabolism, glycogen synthase (GS) and glycogen phosphorylase (GP), were analyzed. When exposed to anoxia, the nervous ganglia of the snail achieved a sustained glucose uptake and glycogen synthesis levels, which seems important to maintain neural homeostasis. However, the activities of GS and GP were reduced, indicating a possible metabolic depression in the CNS. During the aerobic recovery period, the enzyme activities returned to basal values. The possible strategies used by Megalobulimus abbreviatus CNS to survive anoxia are discussed.