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B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 3, e58651- p.Article in journal (Refereed) Published
Abstract [en]

The B(0)AT2 protein is a product of the SLC6A15 gene belonging to the SLC6 subfamily and has been shown to be a transporter of essential branched-chain amino acids. We aimed to further characterize the B(0)AT2 transporter in CNS, and to use Slc6a15 knock out (KO) mice to investigate whether B(0)AT2 is important for mediating the anorexigenic effect of leucine. We used the Slc6a15 KO mice to investigate the role of B(0)AT2 in brain in response to leucine and in particular the effect on food intake. Slc6a15 KO mice show lower reduction of food intake as well as lower neuronal activation in the ventromedial hypothalamic nucleus (VMH) in response to leucine injections compared to wild type mice. We also used RT-PCR on rat tissues, in situ hybridization and immunohistochemistry on mouse CNS tissues to document in detail the distribution of SLC6A15 on gene and protein levels. We showed that B(0)AT2 immunoreactivity is mainly neuronal, including localization in many GABAergic neurons and spinal cord motor neurons. B(0)AT2 immunoreactivity was also found in astrocytes close to ventricles, and co-localized with cytokeratin and diazepam binding inhibitor (DBI) in epithelial cells of the choroid plexus. The data suggest that B(0)AT2 play a role in leucine homeostasis in the brain.

Place, publisher, year, edition, pages
2013. Vol. 8, no 3, e58651- p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-199769DOI: 10.1371/journal.pone.0058651ISI: 000318334500110PubMedID: 23505546OAI: oai:DiVA.org:uu-199769DiVA: diva2:621136
Available from: 2013-05-13 Created: 2013-05-13 Last updated: 2017-12-06Bibliographically approved
In thesis
1. Characterization of Amino Acid Transporters in the Brain: Molecular and Functional Studies of Members within the Solute Carrier Families SLC38 and SLC6
Open this publication in new window or tab >>Characterization of Amino Acid Transporters in the Brain: Molecular and Functional Studies of Members within the Solute Carrier Families SLC38 and SLC6
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Solute carriers (SLCs) comprise the largest group of transporters in humans and there are currently 52 SLC families. They are embedded in cellular membranes and transport numerous molecules; defects in many of the genes encoding SLCs have been connected to pathological conditions, and several SLCs are potential drug targets.

The SLC38 family has in total eleven members in humans and they encode transporters called SNATs. In paper I and paper II, we reported molecular and functional characterization of Slc38a7 and Slc38a8, two of the previous orphan members in the family which we suggested to be named SNAT7 and SNAT8, respectively. Using in situ hybridization and immunohistochemistry, these transporters showed similar expression pattern and localized to neurons in the brain For functional characterization proteins were overexpressed in X. laevis oocytes and an Uptake Assay and electrophysiological recordings showed preferred transport of L-glutamine, L-histidine, L-alanine, L-asparagine, L-aspartate and L-arginine for SNAT7. A similar pattern was seen for SNAT8 in a slightly different order of affinities. We classified SNAT7 as a system N transporter and SNAT8 as belonging to system A, and suggests that SNAT7 and SNAT8 could play a role in the glutamine/glutamate(GABA) cycle (GGC) in the brain.

Furthermore, we studied the vesicular B0AT3 (Slc6a17) transporter in paper III, and the sodium-coupled amino acid transporter B0AT2 (Slc6a15) in paper IV. Tissue expression studies showed similar localization of Slc6a17 and Slc6a15 mRNA using in situ hybridization and real-time PCR. In paper III, vesicular localization of B0AT2 was shown in both excitatory and inhibitory neurons. When challenging the monoaminergic system with drugs both Slc6a17 and Slc6a15 were upregulated. Suggested roles for the transporters are thereby in synaptic remodeling by regulating the availability of free amino acids used as precursors needed in neurotransmitter synthesis. Moreover, in paper IV, immunohistochemistry showed B0AT3 localization to neurons, astrocytes and epithelial cells of the choroid plexus. Leucine injections caused a smaller reduction of food intake as well as higher neuronal activation in the paraventricular hypothalamic nucleus in Slc6a15 KO mice, compared with wild type mice. This suggests B0AT2 involvement in the anorexigenic effects of leucine.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 960
Keyword
Amino acid transporter, Solute Carrier, Glutamine, Leucine, SNAT, B0AT
National Category
Neurosciences
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-212610 (URN)978-91-554-8832-1 (ISBN)
Public defence
2014-02-14, B/B22, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-01-23 Created: 2013-12-12 Last updated: 2014-02-10
2. Characterization of Centrally Expressed Solute Carriers: Histological and Functional Studies with Transgenic Mice
Open this publication in new window or tab >>Characterization of Centrally Expressed Solute Carriers: Histological and Functional Studies with Transgenic Mice
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[en]
: His
Abstract [en]

The Solute Carrier (SLC) superfamily is the largest group of membrane-bound transporters, currently with 456 transporters in 52 families. Much remains unknown about the tissue distribution and function of many of these transporters. The aim of this thesis was to characterize select SLCs with emphasis on tissue distribution, cellular localization, and function.       In paper I, we studied the leucine transporter B0AT2 (Slc6a15). Localization of B0AT2 and Slc6a15 in mouse brain was determined using in situ hybridization (ISH) and immunohistochemistry (IHC), localizing it to neurons, epithelial cells, and astrocytes. Furthermore, we observed a lower reduction of food intake in Slc6a15 knockout mice (KO) upon intraperitoneal injections with leucine, suggesting B0AT2 is involved in mediating the anorexigenic effects of leucine.     In paper II, we studied the postnatal, forebrain-specific deletion of Slcz1, belonging to the SLC18 family, in conditional KO mice (cKO). We observed a decreased response to diazepam and a higher neuronal activity in cortex and hippocampus of cKO mice, as well as an impairment in short-term recognition memory. Intracellular expression was found in neurons but not astrocytes with IHC, indicating SLCZ1 is implicated in neuronal regulation of locomotion and memory.    In paper III, we performed the first detailed histological analysis of PAT4, a transporter belonging to the SLC36 family, involved in the activation of mTOR complex 1 on lysosomes. We found abundant Slc36a4 mRNA and PAT4 expression in mouse brain, using ISH and IHC. We used IHC to localize PAT4 to both inhibitory and excitatory neurons and epithelial cells. We also found both intracellular- and plasmalemmal expression and partial colocalization of PAT4 with lysosomal markers.    Lastly, in paper IV, we provided the first tissue mapping of orphan transporter MCT14 (SLC16A14). Using qPCR, we detected moderate to high Slc16a14 mRNA in the central nervous system and kidney. We found widespread Slc16a14 and MCT14 in mouse brain using ISH and IHC. We also found MCT14 to have intracellular and plasmalemmal expression in mainly excitatory but also inhibitory neurons, as well as epithelial cells. We found MCT14 to be most closely related to MCT8, MCT2 and MCT9, suggesting a similar role for this transporter.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1215
Keyword
SLC, Solute carriers, Amino acid transporter, PAT, B0AT2, mTORC1
National Category
Neurosciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-282956 (URN)978-91-554-9555-8 (ISBN)
Public defence
2016-06-03, B:21, Husargatan. 75124 Uppsala, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-05-12 Created: 2016-04-08 Last updated: 2016-06-01

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Hägglund, Maria G ARoshanbin, SaharHellsten, Sofie VTodkar, AniruddhaSchiöth, Helgi BFredriksson, Robert

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