We found out high expression of the gene in hypothalamic areas such as SON, VMH, AH, PVN, and in Arc. wire 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. Introduction The essential amino acids leucine and valine cannot be synthesized and must consequently be taken up from the diet. Dietary amino acids can enter the bloodstream and therefore reach the brain through mechanisms that include uptake by amino acid transporters [1]C[3], where they may be utilized for energy, in biosyntheses of additional molecules and also as direct regulators of mind functions. Dietary leucine offers been shown to decrease diet-induced obesity [4] and to activate the mammalian target of rapamycin (mTOR) signalling and to decrease food intake and body weight [5]. The SLC6A15 transporter was recognized in 1992 by Uhl hybridization data [13]C[15]. The SLC6A15 transporter has been functionally characterized like a Na+-coupled amino acid transporter. B0AT2 mediates transport of a broad range of amino acids, showing high-affinity for methionine, proline, and the branched-chain amino acids (BCAAs) valine, leucine and isoleucine [9], [12]. Although B0AT2 is fairly well characterized in the biochemical level, the physiological relevance of this transporter is unfamiliar. knockout (KO) mice, originally termed v7-3 KO mice [11], are viable, fertile and display behaviours much like those of crazy type (WT) mice. KO mice experienced however a 40% reduction in Na+-dependent uptake of leucine, and a 15% reduction in uptake of proline into mind synaptosomes compared to WT mice [11]. Initial work with these KO mice offered support for functions for SLC6A15 in mediating effects of leucine in regulating hunger and food preferences (J. Drgonova, F. Hall, G. Uhl, unpublished observations). Here we used KO mice to investigate the part of B0AT2 in mind in response to leucine and in particular the effect on food intake. KO mice showed reduced reduction of food intake and lower neuronal activation in the ventromedial hypothalamic nucleus (VMH) in response to leucine injections compared to WT mice. We display that B0AT2 mRNA and protein are abundant in neurons and astrocytes in hypothalamus and in additional sites Cobimetinib hemifumarate of the brain, using immunohistochemistry and hybridization. B0AT2 immunoreactivity also co-localizes with diazepam binding inhibitor (DBI) and cytokeratin in epithelial cells of the choroid plexus. Materials and Methods Honest statement All animal procedures were authorized by the local honest committee in Uppsala Mmp12 and adopted Cobimetinib hemifumarate the guidelines of European Areas Council Directive (86/609/EEC). Western blot Antibody specificity We performed a western blot analysis of B0AT2 on mind tissue from adult, male C57Bl6/J mice (Taconic M&B, Denmark). The cells was homogenized in homogenization buffer (50 mM Tris, 150 mM NaCl, 4 mM MgCl, 0.5 mM EDTA, 2% Triton X-100 and 1mM Protease inhibitor PMSF (Sigma-Aldrich, USA) diluted in isopropanol). Protein concentrations were determined by protein assay DC (Bio-Rad, Hercules, USA) according to the manufacturer’s instructions. Gel electrophoresis was used to separate the protein lysate (50 g and 200 g) together with PageRuler prestained protein ladder (Fermentas, Canada), on a Mini-Protean TGX gel (4C10%, Bio-Rad, Hercules, USA) in operating buffer (0.1% SDS, 0.025 M Tris base and 0.192 M glycine). The proteins were transferred to a Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA) in transfer buffer (0.025 Tris base, 0.192 M glycine and 20% methanol) and pre-blocked for 1 h in blocking buffer (5% non-fat dry milk (Bio-RAD, Hercules, USA) diluted in 0.15 M NaCl, 0.01 M Tris, 0.005% Tween-20, pH 8.0). The membrane was incubated with the custom made polyclonal B0AT2 antibody (directed against Cobimetinib hemifumarate the peptide sequence: (NH2-)DSVEEVSKKSELIVC(-CONH2); Table S1) over night at 4C. After washes in water, the Cobimetinib hemifumarate membrane was incubated for 1 h with horseradish peroxidase conjugated secondary antibody followed by detection with the enhanced chemiluminescent (ECL) method. The membrane was incubated for 3 min inside a 1:1 mixture.
Categories:Potassium Channels, Non-selective