Astrocytes are multifunctional cells of the central nervous system and they play an important role in many physiological and pathological processes in the brain. Astrocytes are ideally positioned in the brain. With their processes, they are simultaneously in contact with the endothelium vessel wall and the adjacent cells. This allows them to take glucose from the blood, metabolize it in the process of aerobic glycolysis to lactate, which is then delivered to neurons. Neurons use lactate as a source of energy. This process is regulated by noradrenergic stimulation from the brain nucleus locus coeruleus. On their membrane astrocytes express adrenergic receptors (ARs) that are coupled with different types of G-proteins. Stimulation of α1- and β-ARs activates the Ca2+- and cAMP-signaling pathways, respectively, what affects a whole series of reactions in the cell, including glucose metabolism. The role of individual either Ca2+- or cAMP-signaling pathway in the regulation of glucose metabolism in astrocytes has not been fully studied yet. The purpose of the thesis was to determine the contribution of individual pathway to the production of lactate in the cell, upon stimulation of α1- and/or β-ARs with selective agonists, phenylephrine (PE) and isoprenaline (Iso), respectively. Using Laconic, a genetically encoded FRET based nanosensor for intracellular L-Lactate measurements, we found that both pathways have approximately the same contribution to the production of lactate following adrenergic stimulation and that there is a synergistic effect between them. Namely, after the exposure of ARs to both agonists at the same time (Iso+PE), we measured higher FRET signal increase rate and FRET amplitude, which was ~5-fold higher than in the control experiments. Using the glycogenolysis inhibitor DAB, it was confirmed that part of glucose after entering the cell is first incorporated into glycogen (so called glycogen shunt) and only then mobilized and metabolized to lactate. Measurements of intracellular glucose levels with FRET nanosensors FLII12PGLU-700μ6, after stimulation of α1-ARs with phenylephrine, in the presence and absence of DAB, showed that the activation of the Ca2+ signalling pathway increases the concentration of cytosolic free glucose. Higher increase in free glucose after α1-AR activation in the presence of DAB was observed, which indicates, that glucose after uptake in cytosol from extracellular space or endoplasmic reticulum, accumulates in the cytosol to a greater extent due to inhibited glycogen shunt.
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