Selective astrocytic gap junctional trafficking of molecules involved in the glycolytic pathway: impact on cellular brain imaging

Abstract

To assess the specificity of metabolite trafficking among gap junction-coupled astrocytes, we developed novel, real-time, single-cell enzymatic fluorescence assays to assay cell-to-cell transfer of unlabeled glycolytic intermediates and report (i) highly restricted transfer of glucose-6-phosphate (P) and two analogs, deoxyglucose (DG)-6-P, and 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG-6-P, compared with DG and 2-and 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-DG, (ii) extensive junctional diffusion of glyceraldehyde-3-P, NADH, and NADPH plus three anionic fluorescent dyes used as internal standards for transfer assays, and (iii) stimulation of gap junctional communication by increased intracellular Na + that also evokes metabolic responses in nearby coupled astrocytes. Thus, dye transfer does not predict gap junctional permeability of endogenous metabolites. Intracellular retention of flux-regulating compounds (e.g. glucose-6-P) may be necessary for local metabolic control, whereas 'syncytial sharing' may dissipate the work load on peri-synaptic astrocytes. Imaging of brain functional activity depends on local accumulation of exogenous or endogenous signals, and DG-6-P is trapped in the cell where it is phosphorylated, whereas rapid dispersal of cytoplasmic NAD(P)H and labeled glucose metabolites throughout the astrocytic syncytium can interfere with cellular assessment of neuron-astrocyte relationships in autoradiographic, fluorescence microscopic, and magnetic resonance spectroscopic studies.

Contributions of glycogen to astrocytic energetics during brain activation

“…Advantages of storing glucose as glycogen include (i) the glucose polymer has low osmotic activity compared with an equivalent number of ‘free’ glucose molecules, (ii) both the synthesis and degradation of glycogen are tightly regulated, (iii) Glc-6-P can be rapidly generated from glycogen without using ATP at the hexokinase step, and (iv) Glc-6-P is a very poor substrate for passage through astrocytic gap junctions (Gandhi et al 2009b), so it serves as fuel for the astrocyte where it was generated and it also regulates hexokinase activity in that same cell. Thus, glycogen synthesized when glucose supply exceeds demand has an energetic advantage for astrocytes during brain activation when demands for glucose and ATP increase.…”

“…The proposed role of Glc-6-P in governing fuel supplies to astrocytes and neurons has an added dimension because this regulatory metabolite is highly restricted from passage through gap junctional pores (Gandhi et al 2009b). Deoxyglucose-6-P and the fluorescent glucose analog, 2-NBDG-6, also have very low gap junctional permeability, confirming the concept that hexose-6-phosphates do not participate in gap junctional communication.…”

“…Deoxyglucose-6-P and the fluorescent glucose analog, 2-NBDG-6, also have very low gap junctional permeability, confirming the concept that hexose-6-phosphates do not participate in gap junctional communication. [ 14 C]Glc-6-P was reported to traverse gap junctions in cultured astrocytes (Tabernero et al 2006; Tabernero et al 1996), but only label transfer was measured, and apparent diffusion of [ 14 C]Glc-6-P through gap junctions must have been due to its metabolism by enzymes released during the scrape-load procedure to produce labeled downstream gap junction-permeant molecules that include glyceraldehyde-6-P and lactate (Gandhi et al 2009b). …”

“…Coupled astrocytes not only regulate their own metabolic activity, they can also share glucose, downstream metabolites, and redox and signaling compounds with other coupled astrocytes. The large syncytium is comprised of as many as 10,000 cells, and it is linked to the vasculature via endfeet (Gandhi et al 2009a; Gandhi et al 2009b; Ball et al 2007). Astrocytes are crossroads of metabolite trafficking to and from blood.…”

Metabonomic study on women of reproductive age treated with nutritional intervention: screening potential biomarkers related to neural tube defects occurrence

“…The significance of glycolysis to neural cells reported by Bolanos was that a nitric oxide-based regulation of balance between glucose consumption through the glycolytic and pentose phosphate pathway controls neuronal survival during oxidative stress (Bolanos et al, 2007). Moreover, the physiological significance of the glycolytic and pentose phosphate pathway is to generate ribose and reduced coenzyme II (NADPH), a key component in cellular anti-oxidant systems as well as a hydrogen donor for many biological reactions in vivo, including the syntheses of fatty acids, cholesterol and steroids (Gandhi et al, 2009). In addition, lactic acid, the end product of the glycolytic pathway, was also found to be significantly increased in the intervention group (p < 0.01).…”