Investigation on the pyruvate uptake, release and metabolism of cultured rat brain astrocytes
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Authors: | Denker, Nadine | Supervisor: | Dringen, Ralf | 1. Expert: | Aldana, Blanca Irene | Experts: | Hattermann-Koch, Kirsten | Abstract: | Astrocytes play a pivotal role in brain metabolism and in neuroprotection. These cells are considered as very glycolytic, consuming large amounts of glucose that is mainly converted into lactate. The α-keto acid pyruvate is a key player of cellular metabolism that links cytosolic and mitochondrial metabolism as the end product of glycolysis. Pyruvate can also be exported from astrocytes and is in brain believed to have neuroprotective functions. In order to elucidate the pathways involved in astrocytic pyruvate metabolism, primary rat astrocyte cultures were used as model systems to investigate pyruvate consumption and release. Of the different substrates tested (pyruvate, lactate, β-hydroxybutyrate, alanine and acetate) that are known to be metabolized in mitochondria, pyruvate was consumed most efficiently by cultured astrocytes incubated in the absence of glucose. Astrocytes exhibited a nearly time-proportional, concentration-dependent consumption of extracellular pyruvate with apparent Michaelis-Menten kinetic [KM = 0.6 ± 0.1 mM, Vmax = 5.1 ± 0.8 nmol/(min x mg protein)]. Lactate and alanine generated and released in pyruvate-fed astrocytes accounted for approximately 60 % and 10 %, respectively, of the pyruvate consumed within 3 h. The presence of AR-C155858, a monocarboxylate transporter 1 (MCT1)-inhibitor, or the application of 10-fold excess of the MCT1 substrates lactate and β-hydroxybutyrate strongly impaired the astrocytic consumption of extracellular pyruvate. Inhibition of the mitochondrial pyruvate carrier (MPC) by UK5099, as well as inhibition of the respiratory chain by the complex III inhibitor Antimycin A also prevented pyruvate consumption. In contrast, BAM15, a mitochondrial uncoupler, strongly accelerated pyruvate consumption in glucose-deprived astrocytes. In the presence of glucose, astrocytes established a transient extracellular steady-state concentration of pyruvate between 150 µM and 300 µM, while lactate in contrast was continuously released and accumulated to millimolar concentrations. In DMEM culture medium, the extracellular pyruvate concentration remained almost constant for days. In amino acid-free incubation buffer, this almost constant extracellular pyruvate level was established within 5 h, with an initial pyruvate release rate of around 60 nmol/(h x mg), and was maintained for several hours. By consumption of excess extracellular pyruvate in the presence of glucose, astrocytes established similar extracellular pyruvate concentrations. Furthermore, pyruvate release was observed in glucose-free incubation buffer after application of mannose, lactate, fructose, sorbitol or alanine. MCT1 inhibition by AR-C155858 decreased the extracellular pyruvate concentration, while MPC inhibition by UK5099 strongly increased the release of glycolytically derived pyruvate. Both antimycin A and BAM15 application resulted in a complete loss of extracellular pyruvate accumulation. The data presented demonstrate that MCT1 is the main transporter involved in pyruvate consumption and release. Modulation of mitochondrial processes revealed a strong involvement of the mitochondrial metabolism in pyruvate utilization. Overall, alterations of pyruvate metabolism presumably modify the intracellular pyruvate concentration, thereby influencing pyruvate consumption and release, as astrocytes seem to establish an equilibrium between their extracellular and intracellular pyruvate concentration. |
Keywords: | pyruvate; astrocytes; glial cells; metabolism; transport; pyruvate carrier; MCT1; glucose metabolism | Issue Date: | 6-Sep-2024 | Type: | Dissertation | DOI: | 10.26092/elib/3308 | URN: | urn:nbn:de:gbv:46-elib82749 | Institution: | Universität Bremen | Faculty: | Fachbereich 02: Biologie/Chemie (FB 02) |
Appears in Collections: | Dissertationen |
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