magistrsko delo
Ena Opačak (Avtor), Marko Kreft (Recenzent), Nina Vardjan (Mentor), Nina Vardjan (Član komisije za zagovor), Marko Kreft (Član komisije za zagovor), Tom Turk (Član komisije za zagovor)

Povzetek

Astrociti so multifunkcionalne celice centralnega živčnega sistema, ki igrajo pomembno vlogo pri številnih fizioloških in patoloških procesih v možganih. Astrociti so v možganih idealno pozicionirani. S svojimi izrastki so hkrati v stiku z endotelijem žil in sosednjimi celicami. To jim omogoča, da privzemajo glukozo iz krvi, jo presnovijo v procesu aerobne glikolize do laktata, ki ga potem posredujejo nevronom. Nevroni laktat porabijo kot vir energije. Ta proces je uravnan z noradrenergično stimulacijo iz možganskega jedra locus coeruleus. Na membrani astrocitov se nahajajo adrenergični receptorji (AR), ki so sklopljeni z različnimi tipi G-proteinov. Po stimulaciji α1- in β-AR pride do aktivacije Ca2+ oz. cAMP signalne poti, kar vpliva na niz reakcij v celici in tudi na metabolizem glukoze. Ker posamezni vpliv Ca2+ oz. cAMP signalne poti na metabolizem glukoze še ni popolnoma raziskan, je bil namen naloge ugotoviti, kakšen je doprinos posamezne poti na nastanek laktata v celici po stimulaciji α1- in β-AR s selektivnimi agonisti teh receptorjev, fenilefrinom (PE) in izoprenalinom (Iso). Z uporabo Laconica, FRET-nanosenzorja za meritve L-laktata, smo ugotovili, da imata obe poti približno enak doprinos k nastanku laktata v celici po adrenergični stimulaciji, prav tako tudi, da obstaja sinergijski učinek med njima. Po izpostavitvi AR obema agonistoma hkrati (Iso+PE) smo namreč zabeležili najvišjo hitrost porasta signala in amplitudo, ki je 5-krat večja glede na kontrolne poskuse. Z uporabo inhibitorja glikogenolize DAB smo potrdili, da se del glukoze po vstopu v celico najprej vgradi v glikogen (t.i. glikogenski obvod) in šele potem sprosti in metabolizira do laktata. Z meritvami koncentracije glukoze v celici s FRET-nanosenzorjem FLII12PGLU-700μ6 po stimulaciji α1-AR s fenilefrinom v prisotnosti in odsotnosti DAB smo pokazali, da aktivacija Ca2+ signalne poti dvigne koncentracijo citosolne glukoze. Večji porast v prosti glukozi po aktivaciji α1-AR smo zabeležili ob inhibiciji glikogenolize, kar nakazuje, da se je glukoza po vstopu v citosol iz zunajceličnine ali endoplazemskega retikuluma zaradi inhibiranega glikogenskega obvoda v večji meri kopičila v citosolu.

Ključne besede

astrociti;adrenergični receptorji;metabolizem;laktat;FRET;

Podatki

Jezik: Slovenski jezik
Leto izida:
Tipologija: 2.09 - Magistrsko delo
Organizacija: UL BF - Biotehniška fakulteta
Založnik: [E. Opačak]
UDK: 577(043.2)
COBISS: 4988751 Povezava se bo odprla v novem oknu
Št. ogledov: 709
Št. prenosov: 270
Ocena: 0 (0 glasov)
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Ostali podatki

Sekundarni jezik: Angleški jezik
Sekundarni naslov: Effects of cAMP and calcium on glucose metabolism in cultured rat astrocytes upon adrenergic receptors activation
Sekundarni povzetek: 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-700μ6, 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.
Sekundarne ključne besede: astrocytes;adrenergic receptors;metabolism;lactate;FRET;
Vrsta dela (COBISS): Magistrsko delo/naloga
Študijski program: 0
Komentar na gradivo: Univ. Ljubljana, Biotehniška fak.
Strani: XI, 59, [1] f.
ID: 11009814