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Solute Carriers in Metabolism Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1380 Solute Carriers in Metabolism Regulation of known and putative solute carriers in the central nervous system EMILIA LEKHOLM ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0104-4 UPPSALA urn:nbn:se:uu:diva-331328 2017 Dissertation presented at Uppsala University to be publicly examined in B21, Biomedicinskt Centrum (BMC), Husargatan 3, Uppsala, Thursday, 30 November 2017 at 09:30 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Docent David Drew (Stockholms Universitet, Department of Biochemistry and Biophysics). Abstract Lekholm, E. 2017. Solute Carriers in Metabolism. Regulation of known and putative solute carriers in the central nervous system. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1380. 44 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0104-4. Solute carriers (SLCs) are membrane-bound transporter proteins, important for nutrient, ion, drug and metabolite transport across membranes. A quarter of the human genome codes for membrane-bound proteins, and SLCs make up the largest group of transporter proteins. Due to their ability to transport a large repertoire of substances across, not just the plasma membrane, but also the membrane of internal organelles, they hold a key position in maintaining homeostasis affecting metabolic pathways. Unfortunately, some of the more than 400 identified SLCs are still not fully characterized, even though a quarter of these are associated with human disease. In addition, there are about 30 membrane-bound proteins with strong resemblance to SLCs, of which very little is known. The aim of this thesis is to characterize some of these putative SLCs, focusing on their localization and function in the central nervous system. Since many of the known SLCs play a vital part in metabolism and related pathways, the response to different nutritional conditions has been used as a key method. MFSD14A and MFSD14B, characterized in Paper I, are putative SLCs belonging to the Major Facilitator Superfamily (MFS) and found to be neuronal, differentially expressed in the mouse central nervous system and transiently upregulated in mouse embryonic cortex cultures due to amino acid deprivation. They were also altered in areas of the mouse brain after starvation as well as after high fat diet. In Paper II, the effect on gene regulation due to complete amino acid starvation was monitored in a mouse hypothalamic cell line and 47 different genes belonging to SLCs, or putative SLCs, were found to be affected. Of these, 15 genes belonged to already known amino acid transporters, whereas 32 were putative SLCs with no known function or SLCs not known to react to amino acids. The three SV2 proteins, SV2A, SV2B and SV2C, were studied in Paper III using human neuroblastoma cell lines. The high metabolic state of cancers often result in an upregulation and alteration of transporter proteins, and alterations of the SV2 proteins were found following different treatments performed in this study. Paper IV focused on putative SLCs of MFS type and their role in glucose metabolism. Mouse embryonic cortex cultures were subjected to glucose starvation and the gene expression of 19 putative transporters were analyzed. All but four of the putative transporters were affected either at 3h or 12h of glucose deprivation. In conclusion, several SLCs and putative SLCs studied in this thesis are strongly affected by alteration in metabolism, either due to amino acids or glucose or both. This makes the putative SLCs dynamic membrane-bound proteins, possibly transporters, highly affected by nutritional status and most likely regulated to maintain homeostasis. Keywords: Solute Carriers, transporter, amino acid starvation, glucose metabolism, MFS, SV2 Emilia Lekholm, Department of Neuroscience, Functional Pharmacology, Box 593, Uppsala University, SE-75124 Uppsala, Sweden. Department of Pharmaceutical Biosciences, Box 591, Uppsala University, SE-75124 Uppsala, Sweden. © Emilia Lekholm 2017 ISSN 1651-6206 ISBN 978-91-513-0104-4 urn:nbn:se:uu:diva-331328 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-331328) To my fantastic family List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Lekholm, E., Perland, E., Eriksson, M.M., Hellsten, S.V., Lindberg, F.A., Rostami, J., Fredriksson, R. (2017) Putative membrane-bound transporter MFSD14A and MFSD14B are neuronal and affected by nutrient availability. Front Mol Neu- roscience, 10(11) II Hellsten, S.V., Lekholm, E., Ahmad, T., Fredriksson, R. (2017) The gene expression of numerous SLC transporters is altered in the immortalized hypothalamic cell line N25/2 following amino acid starvation. FEBS Open bio, 7(2):249–264 III Lekholm, E., Ceder, M.M., Forsberg, E., Schiöth, H., Fredriks- son, R. (2017) Treatment of human neuroblastoma cell lines with growth supplements, ATRA or VIP alters synaptic vesicle glycoprotein 2 (SV2) expression. Submitted Manuscript IV Lekholm, E*., Ceder, M.M*., Klaesson, A., Perland, E., Schweizer, N., Fredriksson, R. (2017) Glucose deprived mouse embryonal cortex cultures respond by altering MFS transporter expression and localization. Manuscript *Equal contribution Reprints were made with permission from the respective publishers. Additional Papers V Perland, E., Lekholm, E., Eriksson, M.M., Bagchi, S., Arapi, V., and Fredriksson, R. (2016) The putative SLC yransporters Mfsd5 and Mfsd11 are abundantly expressed in the mouse brain and have a potential role in energy homeostasis. PloS one, 11(6):e0156912 VI Roshanbin, S., Lindberg, F.A., Lekholm, E., Eriksson, M.M., Perland, E., Ahlund, J., Raine, A., Fredriksson, R. (2016) Histo- logical characterization of orphan transporter MCT14 (SLC16A14) shows abundant expression in mouse CNS and kidney. BMC neuroscience 17(1):43 VII Perland, E*., Hellsten, S.V*., Lekholm, E., Eriksson, M.M., Arapi, V. and Fredriksson, R. (2017) The novel membrane- bound proteins MFSD1 and MFSD3 are putative SLC trans- porters affected by altered nutrient intake. J Mol Neurosci. 61(2):199-214 VIII Hellsten, S.V., Eriksson, M.M., Lekholm, E., Arapi, V., Per- land, E., Fredriksson, R. (2017) The gene expression of the neu- ronal protein, SLC38A9, is altered in mouse brain following in vivo starvation and high-fat diet. Plos One, 12(2):e0172917 IX Hoeber, J*., Köning, N*., Trolle, C#., Lekholm, E#., Zhou, C., Pankratova, S., Åkesson, E., Fredriksson, R., Aldskogius, H., Kozlova E.N. (2017) A combinatorial approach to induce sen- sory axon regeneration into the dorsal root avulsed spinal cord. Stem Cells Dev, 26(14):1065-1077 X Ceder, M.M., Lekholm, E., Hellsten, S.V., Perland, E., Fred- riksson, R. (2017) The putative membrane-bound protein UNC93A responds highly to starvation in mice. Accepted in Front Mol Neuroscience * And # equal contribution Contents Introduction ................................................................................................... 11 Solute Carriers .......................................................................................... 12 Localization and Regulation of Solute Carriers ....................................... 16 Solute Carriers and Metabolism ............................................................... 17 Solute Carriers in Disease ........................................................................ 19 Aims .............................................................................................................. 22 Paper I ...................................................................................................... 22 Paper II ..................................................................................................... 22 Paper III .................................................................................................... 23 Paper IV ................................................................................................... 23 Methods ........................................................................................................ 24 Homology and Phylogenetic Analysis ..................................................... 24 Gene Expression Analysis ........................................................................ 24 Protein expression .................................................................................... 25 Immunostaining ................................................................................... 25 Enzyme-linked Immunosorbent Assay ................................................ 26 Metabolism Experiments .......................................................................... 26 In vitro ................................................................................................. 26 In vivo .................................................................................................. 27 Results Summary .......................................................................................... 28 Paper I ...................................................................................................... 28 Paper II ..................................................................................................... 29 Paper III .................................................................................................... 30 Paper IV ..................................................................................................
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