title: System N transporters are critical for glutamine release and modulate metabolic fluxes of glucose and acetate in cultured cortical astrocytes: changes induced by ammonia.
abstract: Glutamine (Gln) is synthesized in astrocytes from glutamate (Glu) and ammonia, whereupon it can be released to be transferred to neurons. This study evaluated the as yet not definitely established role of the astrocytic Gln transporters SN1 and SN2 (Slc38a3 and Slc38a5 respectively) in Gln release and metabolic fluxes of glucose and acetate, the canonical precursors of Glu. Cultured neocortical astrocytes were grown in the absence or presence of ammonia (5 mM NH4 Cl, 24 h), which deregulates astrocytic metabolism in hyperammonemic encephalopathies. HPLC analyses of cell extracts of SN1/SN2 siRNA-treated (SN1/SN2-) astrocytes revealed a ~ 3.5-fold increase in Gln content and doubling of glutathione, aspartate, alanine and glutamate contents, as compared to SN1/SN2+ astrocytes. Uptake and efflux of preloaded [(3) H]Gln was likewise significantly decreased in SN1/SN2- astrocytes. The atom percent excess (13) C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the SN1/SN2- cells were incubated with [1-(13) C] glucose, while Gln consumption was not changed. No difference was seen in M + 1 values in SN1/SN2- cells incubated with [2-(13) C] acetate, which were not treated with ammonia. In SN1/SN2- astrocytes, the increase in Gln content and the decrease in radiolabeled Gln release upon exposure to ammonia were found abrogated, and glutamate labeling from [2-(13) C]acetate was decreased as compared to SN1/SN2+ astrocytes. The results underscore a profound role of SN1 and/or SN2 in Gln release from astrocytes under physiological conditions, but less so in ammonia-overexposed astrocytes, and appear to manifest dependence of astrocytic glucose metabolism to Glu/Gln on unimpaired SN1/SN2- mediated Gln release from astrocytes. The astrocytic N system transporters SN1 and SN2 show preponderance to mediate glutamine (Gln) efflux. Under hyperammonemic conditions, accumulation of Gln, a direct product of ammonia detoxification, may deregulate astrocytic metabolism and seems to be responsible for astrocytic swelling. This study evaluated not definitely established role of SN1 and SN2 in Gln release and metabolic fluxes of radiolabeled glucose and acetate. Simultaneous silencing of SN1/SN2 transporters increase Gln, glutathione, aspartate, alanine and glutamate contents (Panel B; marked in red) as compare to non-silenced astrocytes (Panel A). The atom percent excess (13) C values (given as M + 1) for alanine, aspartate and glutamate were decreased when the cells with silenced transporters were incubated with [1-(13) C]glucose, whereas no difference was seen in M + 1 values when those cells were incubated with [2-(13) C]acetate. Ammonia abrogated the increase in Gln content and decrease in radiolabeled Gln release in astrocytes with silenced transporters, but caused a decrease in glutamate labeling from [2-(13) C]acetate.
authors.affiliation: Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Zielińska
authors.affiliation: Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Dąbrowska
authors.affiliation: Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.
authors.forename: Mussie Ghezu
authors.initials: MG
authors.lastname: Hadera
authors.affiliation: Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway.|Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Ursula
authors.initials: U
authors.lastname: Sonnewald
authors.affiliation: Department of Neurotoxicology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Jan
authors.initials: J
authors.lastname: Albrecht
mesh_terms: D000085:Acetates / Q000378:metabolism*; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism*; D000641:Ammonia / Q000494:pharmacology*; D000818:Animals; D001253:Astrocytes / Q000187:drug effects*; D002247:Carbon Isotopes / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D002851:Chromatography, High Pressure Liquid; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D018709:Statistics, Nonparametric; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
chemical_list: D000085:Acetates; D026921:Amino Acid Transport Systems, Neutral; D002247:Carbon Isotopes; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C403222:system N protein 1; D005973:Glutamine; D014316:Tritium; D000641:Ammonia; D005947:Glucose
keywords: 13C isotopomers; acetate; glucose; glutamine transport; mass spectrometry; metabolism