Glycogen phosphorylase | EPFL Graph Search

Glycogen phosphorylase is one of the phosphorylase enzymes (). Glycogen phosphorylase catalyzes the rate-limiting step in glycogenolysis in animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic bond. Glycogen phosphorylase is also studied as a model protein regulated by both reversible phosphorylation and allosteric effects. Mechanism Glycogen phosphorylase breaks up glycogen into glucose subunits (see also figure below): (α-1,4 glycogen chain)n + Pi ⇌ (α-1,4 glycogen chain)n-1 + α-D-glucose-1-phosphate. Glycogen is left with one fewer glucose molecule, and the free glucose molecule is in the form of glucose-1-phosphate. In order to be used for metabolism, it must be converted to glucose-6-phosphate by the enzyme phosphoglucomutase. Although the reaction is reversible in vitro, within the cell the enzyme only works in the forward direction as shown below because the concentration of inorganic phosphate is much higher than that of glucose-1-phosp

Ultra high magnetic field for glial contribution into brain metabolism studied by MR spectroscopy and CEST methods for molecular imaging of glycogen

Elise Marie Catherine Vinckenbosch

Magnetic resonance at ultra-high field increases signal and spectral dispersion. In this thesis, I use those characteristics to investigate three different subjects in 13C spectroscopy, hyperpolarized methods and chemical exchange saturation transfer (CEST) used for molecular imaging. In the brain, metabolism is driven by glia-neuron interactions and is composed of at least two compartments with distinct TCA cycle kinetics. The first study examines cerebral metabolic adaptation after an induced glial TCA-cycle impairment using 1H and 13C spectroscopy. We treated rats with fluoroacetate, which specifically blocks glial aconitase and measured changes in neurochemical profile at euglycemic and hyperglycemic condi-tions by in vivo 1H MR spectroscopy. In addition, we evaluated the brain-compartmentalized metabolic adap-tation by following 13C-incorporation into brain amino acids by modern dynamic 13C MRS methods during infu-sion of [1,6-13C2]glucose. By following [1,6-13C2]glucose incorporation, we successfully observed an augmen-tation of pyruvate carboxylase and glutamine synthetase fluxes and a 20% decrease of glial TCA cycle flux. Neuronal metabolism was also affected. In the second study, we focused on hyperpolarized methods to increase acetate 13C polarization allowing us to follow the kinetics of its metabolic products in the TCA cycle. Indeed, infusion of hyperpolarized [1-13C]acetate, an astrocyte-specific precursor, and the in vivo detection of 2-oxo[5-13C]glutarate (2OG), a TCA intermediate, enables the direct analysis of glial Krebs cycle activity. We examined the effect of hyperpolar-ized acetate concentration on its cerebral metabolism and calculate the production rate of 2OG. A healthy group was compared to rodents treated with fluoroacetate. The conversion rates of acetate to 2OG were calculated and were found to depend on the substrate dose. We successfully estimated 2OG production rates. In partially inhibited TCA cycle conditions, the production rate of 2OG was reduced by a quarter. This level of reduction is agrees with the percentage of inhibition calculated in the first study. Finally, we investigated glycogen imaging in skeletal muscle using the CEST strategy. It has been demonstrat-ed in perfused liver that glycogen can be detected indirectly through the water signal using CEST. Glycogen detection is, due to its resonance close to water, strongly sensitive to spectral dispersion and improved by a higher static magnetic field. In contrast to liver, which contains ~300 mmol/g of glycogen and enjoys good glycogen specificity, myocellular content is also rich in creatine, which produces a spectral signal on the same order of amplitude as its glycogen content of ~80-30 mmol/g. In this study, we allowed glycogen to be de-tectable in vivo and distinguishable from the neighboring creatine at 14.1T. We propose to use creatine as an internal reference for muscular glycoCEST analysis. Scanning muscle after physical exercise, resulted in a 50% reduction of glycoCEST compared to muscle at resting state. This result supportis the specificity of the CEST method and is consistent with literature. This last study demonstrates for the first time that glycoCEST imag-ing is feasible in vivo.

EPFL2018

Disrupting astrocyte-neuron lactate transfer persistently reduces conditioned responses to cocaine

Benjamin Boury-Jamot, Benjamin Boutrel, Anthony Carrard, Pierre Magistretti, Jean-Luc Martin

A central problem in the treatment of drug addiction is the high risk of relapse often precipitated by drug-associated cues. The transfer of glycogen-derived lactate from astrocytes to neurons is required for long-term memory. Whereas blockade of drug memory reconsolidation represents a potential therapeutic strategy, the role of astrocyte-neuron lactate transport in long-term conditioning has received little attention. By infusing an inhibitor of glycogen phosphorylase into the basolateral amygdala of rats, we report that disruption of astrocyte-derived lactate not only transiently impaired the acquisition of a cocaine-induced conditioned place preference but also persistently disrupted an established conditioning. The drug memory was rescued by L-Lactate co-administration through a mechanism requiring the synaptic plasticity-related transcription factor Zif268 and extracellular signal-regulated kinase (ERK) signalling pathway but not the brain-derived neurotrophic factor (Bdnf). The long-term amnesia induced by glycogenolysis inhibition and the concomitant decreased expression of phospho-ERK were both restored with L-Lactate co-administration. These findings reveal a critical role for astrocyte-derived lactate in positive memory formation and highlight a novel amygdala-dependent reconsolidation process, whose disruption may offer a novel therapeutic target to reduce the long-lasting conditioned responses to cocaine.

Nature Publishing Group2015

In vivo magnetic resonance studies of glycogen and hyperpolarized lithium in the rat brain

Ruud van Heeswijk

Nuclear magnetic resonance (NMR) is used for a large array of applications, ranging from chemical characterization to oil drilling to medical imaging. In these fields NMR is used as an investigational tool, but new techniques and applications are continuously being developed as well. The latter is also the subject of this thesis. After an introduction to NMR theory, it focuses on the development of new methodologies for two separate areas of investigation: the metabolism of brain glycogen and hyperpolarization via dynamic nuclear polarization (DNP). Brain glycogen is the main storage form of glucose in the brain. However, because it is present in much lower concentrations in the brain than in other tissues, the significance of this storage and other roles are subject to debate. To better understand the roles of brain glycogen, it is important to quantify its metabolic parameters. The only currently available method to detect brain glycogen in vivo is 13C NMR spectroscopy. Incorporation of 13C-labeled glucose is necessary to allow glycogen measurement, but might be affected by changes in the turnover between glucose and glycogen. We therefore established a protocol to measure the glycogen absolute concentration in the rat brain by eliminating label turnover as a variable. The approach is based on establishing an increased, constant 13C isotopic enrichment (IE). 13C-glucose infusion is then performed at this IE of brain glycogen. As glycogen IE cannot be assessed in vivo, we validated that it can be inferred from the N-acetyl-aspartate (NAA) IE in vivo: after [1-13C]-glucose ingestion, glycogen IE was 2.2 ± 0.1 times that of NAA. Glycogen concentration measured in vivo by 13C NMR (5.8 ± 0.7 µmol/g) was in excellent agreement with post-mortem biochemistry (6.4 ± 0.6 µmol/g). A second glycogen NMR protocol was then implemented to measure concentration and turnover simultaneously. After reaching isotopic steady state for glycogen C1 using [1-13C]-glucose administration, [1,6-13C2]-glucose was infused such that isotopic steady state was maintained at the C1 position, but the C6 position reflected 13C label incorporation. To overcome the large chemical shift displacement error between the C1 and C6 resonances of glycogen, 2D gradient-based localization using the Fourier series window approach was implemented. The glycogen concentration of 5.1 ± 1.6 µmol/g measured from the C1 position was in excellent agreement with concomitant biochemical determinations, while glycogen turnover measured from the rate of label incorporation into the C6 position of glycogen in the α-chloralose anesthetized rat was 0.7 µmol/g/h. The contrast agent Omniscan was added to a formic acid-filled glass reference sphere in the abovementioned protocols in order to shorten its T1 relaxation time and to accelerate the calibration procedure it is used in; therefore it was established that Omniscan in pure 13C formic acid has a relaxivity of 2.9 mM-1s-1. The study was expanded, and the relaxivity of several commercially available gadolinium (Gd)-based contrast agents was determined for X-nuclei resonances with long intrinsic relaxation times. The relaxivity of Omniscan on the glutamate C1 and C5 in aqueous solution was ~ 0.5 mM-1s-1. These relaxivities allow the preparation of solutions with a predetermined short 13C T1. Another isotope with a long relaxation time is lithium-6 (6Li). Lithium is an ion that is widely used in psychotherapy, and 6Li has an intrinsic relaxation time on the order of several minutes, which was strongly affected by the contrast agents. Its relaxivity ranged from ~ 0.1 mM-1s-1 for Omniscan to 0.3 for Magnevist, whereas the relaxivity of Gd-DOTP was 11 mM-1s-1. Overall, these experiments suggested that the presence of submicromolar contrast agent concentrations should be detectable, provided sufficient sensitivity is available, such as that afforded by hyperpolarization. This submicromolar contrast agent concentration detection was demonstrated in a subsequent study: first it was established that lithium-6 can be readily hyperpolarized through DNP within 30 min while retaining a long polarization relaxation time on the order of a minute. The shortening of this relaxation time was used for the detection of 500 nM contrast agent in vitro. Hyperpolarized lithium-6 was then administered to the rat, where its signal retained a relaxation time on the order of 70 s in vivo. Localization experiments implied that the lithium signal originated from within the brain and that it was detectable up to 5 min after administration. Next, ~ 1.5 µM of contrast agent was injected in the rat, and its effect on previously injected hyperpolarized lithium-6 was successfully quantified. It was concluded that the detection of submicromolar contrast agent concentrations using hyperpolarized NMR nuclei such as 6Li may provide a novel avenue for molecular imaging. In a separate set of of experiments, hyperpolarization through DNP was applied to 15N-labeled choline, 13C-labeled bicarbonate and 13C-labeled acetate. All three metabolites were successfully polarized and were detected both in vitro and in vivo.

EPFL2009