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Meritve znotrajcelične signalizacije in presnove v podganjih astrocitih v kulturi po stimulaciji z agonisti receptorjev prostih maščobnih kislin GPR40 in GPR120
ID Tivadar, Nika (Author), ID Vardjan, Nina (Mentor) More about this mentor... This link opens in a new window

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Abstract
Astrociti so celice nevroglije v osrednjem živčnem sistemu, ki se nahajajo med kapilarami in nevroni. Preko glukoznih transporterjev iz krvi privzemajo glukozo, ki se v astrocitih skladišči v obliki glikogena in ob povečani možganski aktivnosti v procesu aerobne glikolize razgradi do laktata. Ta se transportira v nevrone preko laktatnih transporterjev, kjer se uporabi kot vir energije. Na površini astrocitov se izražajo z G-proteini sklopljeni receptorji (GPCR), ki ob aktivaciji sprožijo znotrajcelični Ca2+- in cAMP-signalni poti. Le-ti uravnavata glukozno presnovo v astrocitih. Receptorja GPR120/FFAR4 in GPR40/FFAR1 sta GPCR, ki ju aktivirajo dolgoverižne proste maščobne kisline. Njuna vloga v možganih je slabo raziskana. Zanimalo nas je, kako selektivna agonista GPR120/FFAR4, TUG-891 in GPR40/FFAR1, TAK-875, vplivata na znotrajcelično raven Ca2+ in cAMP ter na glukozno presnovo v astrocitih. S pomočjo Ca2+-indikatorja Fluo-4 AM in FRET-nanosenzorjev za cAMP, glukozo in laktat ter konfokalne mikroskopije v realnem času smo spremljali spremembe v znotrajcelični koncentraciji Ca2+, cAMP, glukoze in laktata v podganjih kortikalnih astrocitih v kulturi po draženju s selektivnima agonistoma TUG-891 ali TAK-875. Draženje astrocitov z 10 μM TUG-891 ni povzročilo sprememb v znotrajcelični koncentraciji Ca2+, a je sprožilo porast v koncentraciji cAMP in laktata, kar nakazuje, na sklopitev GPR120/FFAR4 z Gs-proteinom ter aktivacijo cAMP-signalne poti in aerobne glikolize. Draženje z 10 μM TUG-891 ali TAK-875 ni vplivalo na znotrajcelično raven proste glukoze. Sklepamo, da agonista ne vplivata na privzem glukoze iz zunajceličnine, ki je odvisen od znotrajcelične koncentracije Ca2+, na katero agonista ne vplivata.

Language:Slovenian
Keywords:Ca2+, cAMP, FRET, glukoza, laktat, nanosenzor, TAK-875, Tapsigargin, TUG-891
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Publisher:[N. Tivadar]
Year:2023
PID:20.500.12556/RUL-152591 This link opens in a new window
UDC:577.2(043.2)
COBISS.SI-ID:174465795 This link opens in a new window
Publication date in RUL:30.11.2023
Views:946
Downloads:71
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Secondary language

Language:English
Title:Measurements of intracellular signalling and metabolism in rat cultured astrocytes upon stimulation with agonists of free fatty acid receptors GPR40 and GPR120
Abstract:
Astrocytes are type of neuroglial cells in the central nervous system, which are positioned between capillaries and neurons. They can uptake glucose from blood through glucose transporters. Glucose can be stored in astrocytes in glycogen and during intense brain activity metabolised to lactate in the process of aerobic glycolysis. Neurons can uptake lactate via lactate transporters and use it as an energy source. Astrocytes express G-protein coupled receptors (GPCR) on their surface, which upon activation trigger intracellular Ca2+ and cAMP signaling pathways. The latter regulate glucose metabolism in astrocytes. GPR120/FFAR4 and GPR40/FFAR1 are GPCRs, activated by long chain free fatty acids. Their role in brain is poorly understood. Our aim was to study the effect of selective agonists of GPR120/FFAR4, TUG-891, and GPR40/FFAR1, TAK-875, on intracellular signalling pathways of Ca2+ and cAMP and glucose metabolism in astrocytes. Using fluorescent Ca2+-indicator Fluo-4 AM and FRET-nanosensors in combination with real time confocal microscopy, we monitored changes in intracellular level of Ca2+, cAMP, glucose and lactate in cultured rat cortical astrocytes upon stimulation with agonists TUG-891 or TAK-875. Stimulation with 10 μM TUG-891 had no effect on intracellular Ca2+ concentration, but caused a rise in concentration of cAMP and lactate, which indicates coupling of GPR120/FFAR4 with Gs-protein and activation of cAMP signaling pathway and aerobic glycolysis. Stimulation with 10 μM TUG-891 or TAK-875 had no effect on intracellular free glucose level. We assume that agonists do not affect uptake of extracellular glucose, which depends on intracellular Ca2+-concentration, on which agonists had no effect.

Keywords:Ca2+, cAMP, FRET, glucose, lactate, nanosensor, TAK-875, Tapsigargin, TUG-891

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