Characterization of the transporterB0AT3 (Slc6a17) in the rodent central nervous system.

BACKGROUND: The vesicular B0AT3 transporter (SLC6A17), one of the members of the SLC6 family, is a transporter for neutral amino acids and is exclusively expressed in brain. Here we provide a comprehensive expression profile of B0AT3 in mouse brain using in situ hybridization and immunohistochemistry. RESULTS: We confirmed previous expression data from rat brain and used a novel custom made antibody to obtain detailed co-labelling with several cell type specific markers. B0AT3 was highly expressed in both inhibitory and excitatory neurons. The B0AT3 expression was highly overlapping with those of vesicular glutamate transporter 2 (VGLUT2) and vesicular glutamate transporter 1 (VGLUT1). We also show here that Slc6a17mRNA is up-regulated in animals subjected to short term food deprivation as well as animals treated with the serotonin reuptake inhibitor fluoxetine and the dopamine/noradrenaline reuptake inhibitor bupropion. CONCLUSIONS: This suggests that the B0AT3 transporter have a role in regulation of monoaminergic as well as glutamatergic synapses.

Comprehensive analysis of localization of 78 solute carrier genes throughout the subsections of the rat gastrointestinal tract.

Solute carriers (SLCs), the second largest super-family of membrane proteins in the human genome, transport amino acids, sugars, fatty acids, inorganic ions, essential metals and drugs over membranes. To date no study has provided a comprehensive analysis of SLC localization along the entire GI tract. The aim of the present study was to provide a comprehensive, segment-specific description of the localization of SLC genes along the rat GI tract by employing bioinformatics and molecular biology methods. The Unigene database was screened for rat SLC entries in the intestinal tissue. Using qPCR we measured expression of the annotated genes in the GI tract divided into the following segments: the esophagus, the corpus and the antrum of the stomach, the proximal and distal parts of the duodenum, ileum, jejunum and colon, and the cecum. Our Unigene-derived gene pool was expanded with data from in-house tissue panels and a literature search. We found 44 out of 78 (56%) of gut SLC transcripts to be expressed in all GI tract segments, whereas the majority of remaining SLCs were detected in more than five segments. SLCs are predominantly expressed in gut regions with absorptive functions although expression was also found in segments unrelated to absorption. The proximal jejunum had the highest number of differentially expressed SLCs. In conclusion, SLCs are a crucial molecular component of the GI tract, with many of them expressed along the entire GI tract. This work presents the first overall road map of localization of transporter genes in the GI tract.

Hypothalamic FTO is associated with the regulation of energy intake not feeding reward.

BACKGROUND: Polymorphism in the FTO gene is strongly associated with obesity, but little is known about the molecular bases of this relationship. We investigated whether hypothalamic FTO is involved in energy-dependent overconsumption of food. We determined FTO mRNA levels in rodent models of short- and long-term intake of palatable fat or sugar, deprivation, diet-induced increase in body weight, baseline preference for fat versus sugar as well as in same-weight animals differing in the inherent propensity to eat calories especially upon availability of diverse diets, using quantitative PCR. FTO gene expression was also studied in organotypic hypothalamic cultures treated with anorexigenic amino acid, leucine. In situ hybridization (ISH) was utilized to study FTO signal in reward- and hunger-related sites, colocalization with anorexigenic oxytocin, and c-Fos immunoreactivity in FTO cells at initiation and termination of a meal. RESULTS: Deprivation upregulated FTO mRNA, while leucine downregulated it. Consumption of palatable diets or macronutrient preference did not affect FTO expression. However, the propensity to ingest more energy without an effect on body weight was associated with lower FTO mRNA levels. We found that 4-fold higher number of FTO cells displayed c-Fos at meal termination as compared to initiation in the paraventricular and arcuate nuclei of re-fed mice. Moreover, ISH showed that FTO is present mainly in hunger-related sites and it shows a high degree of colocalization with anorexigenic oxytocin. CONCLUSION: We conclude that FTO mRNA is present mainly in sites related to hunger/satiation control; changes in hypothalamic FTO expression are associated with cues related to energy intake rather than feeding reward. In line with that, neurons involved in feeding termination express FTO. Interestingly, baseline FTO expression appears linked not only with energy intake but also energy metabolism.

The obesity gene, FTO, is of ancient origin, up-regulated during food deprivation and expressed in neurons of feeding-related nuclei of the brain.

Gene variants of the FTO (fatso) gene have recently been strongly associated with body mass index and obesity. The FTO gene is well conserved and found in a single copy in vertebrate species including fish and chicken, suggesting that the ancestor of this gene was present 450 million years ago. Surprisingly, the FTO gene is present in two species of algae but not in any other invertebrate species. This could indicate that this gene has undergone a horizontal gene transfer. Quantitative real-time PCR showed that the gene is expressed in many peripheral and central rat tissues. Detailed in situ hybridization analysis in the mouse brain showed abundant expression in feeding-related nuclei of the brainstem and hypothalamus, such as the nucleus of the solitary tract, area postrema, and arcuate, paraventricular, and supraoptic nuclei as well as in the bed nucleus of the stria terminalis. Colabeling showed that the FTO gene is predominantly expressed in neurons, whereas it was virtually not found in astrocytes or glia cells. The FTO was significantly up-regulated (41%) in the hypothalamus of rats after 48-h food deprivation. We also found a strong negative correlation of the FTO expression level with the expression of orexigenic galanin-like peptide, which is mainly synthesized in the arcuate nucleus. These results are consistent with the hypothesis that FTO could participate in the central control of energy homeostasis.

The solute carrier (SLC) complement of the human genome: phylogenetic classification reveals four major families.

Solute carriers (SLCs) is the largest group of transporters, embracing transporters for inorganic ions, amino acids, neurotransmitters, sugars, purines and fatty acids among other substrates. We mined the finished assembly of the human genome using Hidden Markov Models (HMMs) obtaining a total of 384 unique SLC sequences. Detailed clustering and phylogenetic analysis of the entire SLC family showed that 15 of the families place into four large phylogenetic clusters with the largest containing eight SLC families, suggesting that many of the distinct families of SLCs have a common evolutionary origin. This study represents the first overall genomic roadmap of the SLCs providing large sequence sets and clarifies the phylogenetic relationships among the families of the second largest group of membrane proteins.

Expression profile of the entire family of Adhesion G protein-coupled receptors in mouse and rat.

BACKGROUND: The Adhesion G protein-coupled receptors (GPCRs) are membrane-bound receptors with long N termini. This family has 33 members in humans. Several Adhesion GPCRs are known to have important physiological functions in CNS development and immune system response mediated by large cell surface ligands. However, the majority of Adhesion GPCRs are still poorly studied orphans with unknown functions. RESULTS: In this study we performed the extensive tissue localization analysis of the entire Adhesion GPCR family in rat and mouse. By applying the quantitative real-time PCR technique we have produced comparable expression profile for each of the members in the Adhesion family. The results are compared with literature data and data from the Allen Brain Atlas project. Our results suggest that the majority of the Adhesion GPCRs are either expressed in the CNS or ubiquitously. In addition the Adhesion GPCRs from the same phylogenetic group have either predominant CNS or peripheral expression, although each of their expression profile is unique. CONCLUSION: Our findings indicate that many of Adhesion GPCRs are expressed, and most probably, have function in CNS. The related Adhesion GPCRs are well conserved in their structure and interestingly have considerable overlap in their expression profiles, suggesting similarities among the physiological roles for members within many of the phylogenetically related clusters.

The evolutionary history and tissue mapping of amino acid transporters belonging to solute carrier families SLC32, SLC36, and SLC38.

Members of the solute carrier families (SLC) 32, 36, and 38, together also designated the beta-group of SLCs, are known to transport neutral amino acids. In this paper, we show that these three families were present before the split of the animal lineage and that they are likely to share a common decent. We also show that the APF transporters found in plants are most likely homologous to the mammalian beta-group, suggesting that this type of transporters arouse early in the evolution of eukaryotes. We performed detailed tissue expression analysis of all the members of the beta-group in rat and found several examples of highly specific expression patterns, with SLC38A7 being exclusively found in liver, SLC38A5 in blood, and SLC38A4 in muscle and liver. Moreover, we found that SLC38A10 is expressed in several endocrine organs. We also found that SLC38A1 is highly up regulated in the cortex from rats treated with diazepam and that SLC38A2 is significantly down regulated in the same tissue. In addition, we performed a detailed expression analysis of SLC38A1 and SLC38A6 in mouse brain using in situ hybridization, which showed that both these transporters are widely expressed in the brain.

Detection probability models for bacteria, and how to obtain them from heterogeneous spiking data. An application to Bacillus anthracis.

Efficient and correct evaluation of sampling results with respect to hypotheses about the concentration or distribution of bacteria generally requires knowledge about the performance of the detection method. To assess the sensitivity of the detection method an experiment is usually performed where the target matrix is spiked (i.e. artificially contaminated) with different concentrations of the bacteria, followed by analyses of the samples using the pre-enrichment method and the analytical detection method of interest. For safety reasons or because of economic or time limits it is not always possible to perform exactly such an experiment, with the desired number of samples. In this paper, we show how heterogeneous data from diverse sources may be combined within a single model to obtain not only estimates of detection probabilities, but also, crucially, uncertainty estimates. We indicate how such results can then be used to obtain optimal conclusions about presence of bacteria, and illustrate how strongly the sampling results speak in favour of or against contamination. In our example, we consider the case when B. cereus is used as surrogate for B. anthracis, for safety reasons. The statistical modelling of the detection probabilities and of the growth characteristics of the bacteria types is based on data from four experiments where different matrices of food were spiked with B. anthracis or B. cereus and analysed using plate counts and qPCR. We show how flexible and complex Bayesian models, together with inference tools such as OpenBUGS, can be used to merge information about detection probability curves. Two different modelling approaches, differing in whether the pre-enrichment step and the PCR detection step are modelled separately or together, are applied. The relative importance on the detection curves for various existing data sets are evaluated and illustrated.

mRNA GPR162 changes are associated with decreased food intake in rat, and its human genetic variants with impairments in glucose homeostasis in two Swedish cohorts.

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating intercellular interactions of fundamental physiological importance for survival including regulation of food intake, blood pressure, and hormonal sensing signaling, among other roles. Homeostatic alterations in the physiological status of GPCRs are often associated with underlying causes of disease, and to date, several orphan GPCRs are still uncharacterized. Findings from our previous study demonstrate that the Rhodopsin family protein GPR162 is widely expressed in GABAergic as well as other neurons within the mouse hippocampus, whereas extensive expression is observed in hypothalamus, amygdala, and ventral tegmental area, regions strictly interconnected and involved in the regulation of energy homeostasis and hedonic feeding. In this study, we provide a further anatomical characterization of GPR162 in mouse brain via in situ hybridization as well as detailed mRNA expression in a panel of rat tissues complementing a specie-specific mapping of the receptor. We also provide an attempt to demonstrate a functional implication of GPR162 in food intake-related behavior via antisense knockdown studies. Furthermore, we performed human genetic studies in which for the first time, variants of the GPR162 gene were associated with impairments in glucose homeostasis.

B(0)AT2 (SLC6A15) is localized to neurons and astrocytes, and is involved in mediating the effect of leucine in the brain.

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.

Long evolutionary conservation and considerable tissue specificity of several atypical solute carrier transporters.

The superfamily of Solute Carriers (SLCs) has around 384 members in the human genome grouped into at least 48 families. While many of these transporters have been well characterized with established important biological functions, there are few recently identified genes that are not studied regarding tissue distribution or evolutionary origin. Here we study 14 of these recently discovered SLC genes (HIAT1, HIATL1, MFSD1, MFSD5, MFSD6, MFSD9, MFSD10, SLC7A14, SLC7A15, SLC10A6, SLC15A5, SLC16A12, SLC30A10 and SLC21A21) with the purpose to give much better picture over the sequence relationship and tissue expression of the diverse SLC gene family. We used a range of bioinformatic methods to classify each of these genes into the different SLC gene families. We found that 9 of the 14 atypical SLCs are distant members of the Major Facilitator Superfamily (MFS) clan while the others belong to the APC clan, the DMT clan, the CPA_AT clan and the IT clan. We found most of the genes to be highly evolutionary conserved, likely to be present in most bilateral species, except for SLC21A21 that we found only present in mammals. Several of these transporter genes have highly specific tissue expression profile while it is notable that most are expressed in the CNS with the exception of SLC21A21 and SLC15A5. This work provides fundamental information on 14 transporters that previously have not received much attention enabling a more comprehensive view over the SLC superfamily.

The G protein coupled receptor Gpr153 shares common evolutionary origin with Gpr162 and is highly expressed in central regions including the thalamus, cerebellum and the arcuate nucleus.

The Rhodopsin family of G protein coupled receptors (GPCRs) includes the phylogenetic α-group consisting of about 100 human members. The α-group is the only group of GPCRs that has many receptors for biogenic amines which are major drug targets. Several members of this group are orphan receptors and their functions are elusive. In this study we present a detailed phylogenetic and anatomical characterization of the Gpr153 receptor and also attempt to study its functional role. We identified the homologue of Gpr153 in the elephant shark genome and phylogenetic and synteny analyses revealed that Gpr162 and Gpr153 share a common ancestor that split most likely through a duplication event before the divergence of the tetrapods and the teleost lineage. A quantitative real-time PCR study reveals widespread expression of Gpr153 in the central nervous system and all the peripheral tissues investigated. Detailed in situ hybridization on mouse brain showed specifically high expression in the thalamus, cerebellum and the arcuate nucleus. The antisense oligodeoxynucleotide knockdown of Gpr153 caused a slight reduction in food intake and the elevated plus maze test showed significant reduction in the percentage of time spent in the centre square, which points towards a probable role in decision making. This report provides the first detailed characterization of the evolution, expression and primary functional properties of the Gpr153 gene.

C6ORF192 forms a unique evolutionary branch among solute carriers (SLC16, SLC17, and SLC18) and is abundantly expressed in several brain regions.

About one third of all known human proteins are membrane proteins, which constitute several large families. The solute carriers with over 300 known members are probably the second largest family with additional members frequently being identified. We recently found a new putative solute carrier, C6ORF192, belonging to the major facilitator superfamily type of proteins. The gene is evolutionary highly conserved with a single copy present in each of the genomes from mouse, rat, chicken, zebrafish, tetraodon, Caenorhabditis elegans, and Drosophila melanogaster. C6ORF192 forms a novel evolutionary branch of solute carriers and is most closely related to the solute carrier families 16, 17, and 18, all members of the major facilitator superfamily. Ten of the 25 members of these families show amino acid identity with C6ORF192 ranging from 21% to 27%. C6ORF192 differs however, structurally from these families and does not share key motifs in the transmembrane domains. Expression profiling by quantitative real-time polymerase chain reaction and in situ hybridization showed that C6ORF192 transcript can be detected in several tissues, both in the central nervous system and the periphery.