Enterocyte-specific regulation of the apical nutrient transporter SLC6A19 (B0AT1) by transcriptional and epigenetic networks

Enterocytes are specialized to absorb nutrients from the lumen of the small intestine by expressing a select set of genes to maximize the uptake of nutrients. They develop from stem cells in the crypt and differentiate into mature enterocytes while moving along the crypt-villus axis. Using, as an example, the Slc6a19 gene, encoding the neutral amino acid transporter B0AT1, we studied regulation of the gene by transcription factors and epigenetic factors in the intestine. To investigate this question we used a fractionation method to separate mature enterocytes from crypt cells and analysed gene expression. Transcription factors HNF1a and HNF4a activate transcription of the Slc6a19 gene in villus enterocytes, while high levels of SOX9 repress expression in the crypts. CpG dinucleotides in the proximal promoter were highly methylated in the crypt and fully de-methylated in the villus. Furthermore, histone modification H3K27Ac, indicating an active promo! ter, was prevalent in villus cells but barely detectable in crypt cells. The results suggest that Slc6a19 expression in the intestine is regulated at three different levels involving promoter methylation, histone modification and opposing transcription factors

Molecular insights into the regulation of glutamine transport across cellular membranes

Glutamine is the most abundant amino acid in the body. As the primary nitrogen carrier between cells, it plays essential roles in inter-organ nutrition and cellular homeostasis. The transport of glutamine across cellular membranes is facilitated by membrane transporters of the Solute-Linked Carrier (SLC) families. SLC38 is one such family, whose members encode SNAT proteins (Sodium-Neutral Amino acid Transporter or System N/A Transporter), which together transport a broad range of neutral amino acids. Encoded by S/c38a3, SNAT3 prefers glutamine, histidine and asparagine as substrates. Substrate transport can be bidirectional since it is accompanied by Na{u207A} symport and H{u207A} antiport. S/c38a3 has a narrow tissue specificity, and is expressed in the central nervous system, liver, kidney and pancreas. In the central nervous system, glutamine efflux via SNAT3 in astrocytes facilitates neurotransmitter recycling to maintain effective neurotransmission and prevent neurotoxicity. In the liver, glutamine uptake and its subsequent breakdown in hepatocytes facilitates in the detoxification of ammonia by feeding into the urea cycle. In the kidney, as the sole portal for glutamine entry through the basolateral membranes of proximal tubules, SNAT3 is required to maintain acid-base balance, especially during metabolic acidosis. In the pancreas, SNAT3 is thought to facilitate insulin release. Despite being at the start of such key metabolic processes, the molecular mechanisms governing the regulation of SNAT3 is a lesser explored area. This thesis examined the regulation of SNAT3 at an epigenetic, transcriptional, post{u00AD} transcriptional and post-translational level. Firstly, this thesis showed that glutamine uptake via SNAT3 was down-regulated by the Protein Kinase C (PKC). Using Xenopus laevis oocytes, it was shown that the down-regulation occurred in a caveolin-dependent, dynamin-independent manner. Furthermore, the effect of PKCon SNAT3 activity was only observed in hepatocytes, and not in astrocytes. Secondly, in an attempt to understand the causes for the narrow tissue specificity of the gene, the transcriptional regulation of S/c38a3 was studied. Analyses of the S/c38a3 genomic sequence identified the position of the gene core promoter merely 5Obp upstream of the transcriptional start site. Site-directed mutagenesis and chromatin immunoprecipitation demonstrated that transcription was driven by the ubiquitous transcription factor, Specificity Protein 1 (Spl). Bisulfite genome sequencing revealed that four cytosine residues in the proximal promoter region were methylated in the non-S/c38a3 expressing mouse intestine and unmethylated in the mouse liver. Finally, the mechanisms governing the upregulation of S/c38a3 mRNA during metabolic acidosis were examined. The results from this study demonstrated that there may be transcriptional upregulation of the gene during acute acidosis. Additionally, the S/c38a3 3' untranslated region contained elements that destabilized the mRNA during normal conditions, and stabilized it during chronic acidosis. Moreover, these results demonstrated that S/c38a3 was a part of a common regulatory pathway that controlled multiple genes that are upregulated during metabolic acidosis. Taken together, this thesis has shed light to the regulatory events that control glutamine transport via SNAT3 at a molecular level

Rapid downregulation of the rat glutamine transporter SNAT3 by a caveolin-dependent trafficking mechanism in Xenopus laevis oocytes

The glutamine transporter SNAT3 is involved in the uptake and release of glutamine in the brain, liver, and kidney. Substrate transport is accompanied by Na+ cotransport and H+ antiport. In this study, treatment of Xenopus laevis oocytes expressing rat SNAT3 with the phorbol ester PMA resulted in a rapid downregulation of glutamine uptake in less than 20 min. PMA treatment of oocytes coexpressing SNAT3 and the monocarboxylate transporter MCT1 reduced SNAT3 activity only, demonstrating the specificity of the regulatory mechanism. Single or combined mutations of seven putative phosphorylation sites in the SNAT3 sequence did not affect the regulation of SNAT3 by PMA. Expression of an EGFP-SNAT3 fusion protein in oocytes established that the down-regulation was caused by the retrieval of the transporter from the plasma membrane. Coexpression of SNAT3 with dominant-negative mutants of dynamin or caveolin revealed that SNAT3 trafficking occurs in a dynamin-independent manner and is influenced by caveolin. Although system N activity was not affected by PMA in cultured astrocytes, a downregulation was observed in HepG2 cells

Expression of glutamine transporter Slc38a3 (SNAT3) during acidosis is mediated by a different mechanism than tissue-specific expression

Background: Despite homeostatic pH regulation, systemic and cellular pH changes take place and strongly influence metabolic processes. Transcription of the glutamine transporter SNAT3 (Slc38a3) for instance is highly up-regulated in the kidney during metabolic acidosis to provide glutamine for ammonia production. Methods: Slc38a3 promoter activity and messenger RNA stability were measured in cultured cells in response to different extracellular pH values. Results: Up-regulation of SNAT3 mRNA was mediated both by the stabilization of its mRNA and by the up-regulation of gene transcription. Stabilisation of the mRNA involved a pH-response element, while enhanced transcription made use of a second pH-sensitive Sp1 binding site in addition to a constitutive Sp1 binding site. Transcriptional regulation dominated the early response to acidosis, while mRNA stability was more important for chronic adaptation. Tissue-specific expression of SNAT3, by contrast, appeared to be controlled by promoter methylation and histone modifications. Conclusions: Regulation of SNAT3 gene expression by extracellular pH involves post-transcriptional and transcriptional mechanisms, the latter being distinct from the mechanisms that control the tissue-specific expression of the gene.This study was supported by grants from the National Health and Medical Research Council (585479) to S.B. and by the NIH (DK60596 and DK53307) to M.H

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na+ and Cl--dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na+-binding sites and a putative Cl--binding site. Mutation of all residues in the two proposed Na+-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na+-binding site 1) and mutants S20A, S20G, S20G/G405S (Na+-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na+-binding site 1 caused a decrease of the Na+-K0.5, whereas mutations of site 2 increased the K0.5. Mutation of Na+-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates36Cl- demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na+/1Cl--proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria

Sodium translocation by the iminoglycinuria associated imino transporter (SLC6A20)

The system IMINO transporter plays an essential role in the transport of proline and hydroxyproline in the intestine and kidney. Its molecular correlate has been identified and named SIT1 or IMINO (SLC6A20). Initial characterization of the transporter showed it to be Na� and Cl�-dependent, but the stoichiometry remained unresolved. Using homology modeling along the structure of the bacterial leucine transporter LeuT, we identified two highly conserved Na�-binding sites and a putative Cl�-binding site. Mutation of all residues in the two proposed Na�-binding sites revealed that most of them were essential for uptake and completely inactivated the transporter. However, mutants A22V (Na�-binding site 1) and mutants S20A, S20G, S20G/G405S (Na�-binding site 2) were partially active and characterized further. Flux studies suggested that mutations of Na�-binding site 1 caused a decrease of the Na�-K0.5, whereas mutations of site 2 increased the K0.5. Mutation of Na�-binding site 1 also changed the ion selectivity of the IMINO transporter. IMINO actively translocates 36Cl� demonstrating that the proposed chloride binding site is used in the transporter. Accumulation experiments and flux measurements at different holding potentials showed that the transporter can work as a 2Na�/1Cl�- proline cotransporter. The proposed homology model allows to study mutations in IMINO associated with iminoglycinuria