B0AT2 (SLC6A15) Is Localized to Neurons and Astrocytes, and Is Involved in Mediating the Effect of Leucine in the Brain

Abstract

The B0AT2 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 B0AT2 transporter in CNS, and to use Slc6a15 knock out (KO) mice to investigate whether B0AT2 is important for mediating the anorexigenic effect of leucine. We used the Slc6a15 KO mice to investigate the role of B0AT2 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 B0AT2 immunoreactivity is mainly neuronal, including localization in many GABAergic neurons and spinal cord motor neurons. B0AT2 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 B0AT2 play a role in leucine homeostasis in the brain.

Transport of l-Glutamine, l-Alanine, l-Arginine and l-Histidine by the Neuron-Specific Slc38a8 (SNAT8) in CNS

“…Mouse brains were surgically removed from anesthetized adult male C57Bl6/J mice (Taconic M&B, Denmark) as previously described 52 . In short, a Leica VT1000S vibratome (Leica Microsystems, Germany) was used for sectioning 70 µm free floating sections.…”

“…20 Non-fluorescent immunohistochemistry on free floating sections -Single immunohistochemistry was performed with the SNAT8 antibody (diluted 1:5000, Innovagen, Sweden) as previously described 52 .…”

Brain interstitial fluid glutamine homeostasis is controlled by blood–brain barrier SLC7A5/LAT1 amino acid transporter

“…Baseline ratio of hGln/Gln measured by liquid chromatography mass spectrometry (LC-MS) before IP injection was 6.6 AE 2% in plasma and 0.13 AE 0.3% in microdialysates. The higher baseline hGln levels detected in plasma may have been an artifact of the LC-MS analysis since the naturally occurring frequency of the 15 N isotope is 0.1-0.5%. 29 LC-MS may have over-estimated hGln in plasma but not in microdialysis samples.…”

“…The data are based on in vivo and in vitro studies. 2,3,[12][13][14][15][16][17][18][19][20][21][22][23]24 AA content was quantified and AA regulation investigated by in vivo microdialysis in freely moving mice.…”

A novel role for PHT1 in the disposition of l -histidine in brain: In vitro slice and in vivo pharmacokinetic studies in wildtype and Pht1 null mice

“…Studies on the transport of L-His in brain would extend our understanding of neuropeptide regulation, histamine homeostasis, and potential targets for drug delivery to neuronal and non-neuronal cells. In the central nervous system (CNS), major transporters for L-His include the Na + -coupled neutral amino acid transporters (e.g., SNATs, which belong to the SLC38 family) [15,16], Na + -independent amino acid transporters (e.g., LATs and CATs, which belong to the SLC7 family) [17,18], some members of the Na + -and Cl − -dependent neurotransmitter transporter family (e.g., B 0 AT2 and NTT4, which belong to the SLC6 family) [19–21], as well as the peptide/histidine transporters (e.g., PHT1 and PHT2, which belong to the SLC15 family) [4,22]. …”