[VNUT Is a Therapeutic Target for Type 2 Diabetes and NASH].

ATP, used in cells as an energy currency, also acts as an extracellular signaling molecule. Studies of purinergic receptor subtypes have revealed that purinergic chemical transmission plays important roles in various cell types. The vesicular nucleotide transporter (VNUT), the ninth transporter in the SLC17 organic anion transporter family, is essential for vesicular ATP storage and its subsequent release. The VNUT is localized on the membrane of secretory vesicles and actively transports ATP into vesicles using an electrochemical gradient of protons supplied by vacuolar proton ATPase (V-ATPase) as a driving force. ATP acts as a damage-associated molecular pattern (DAMPs), contributing to the persistence of chronic inflammation. Chronic inflammation induces systemic insulin resistance, which is the underlying pathology of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). We previously demonstrated that ATP transported in insulin granules via the VNUT negatively regulates insulin secretion. We also found that hepatocytes release ATP in a VNUT-dependent manner, which elicits hepatic insulin resistance and inflammation. VNUT-knockout mice exhibited improved glucose tolerance and were resistant to the development of high fat diet-induced NAFLD. In this article, we summarize recent advances in our understanding of the mechanism of the VNUT, the development of inhibitors, and its pathological involvement in type 2 diabetes and NAFLD. The pharmacological inhibition of the VNUT may represent a potential therapeutic approach for the treatment of metabolic diseases.

Live Imaging Reveals Cerebellar Neural Stem Cell Dynamics and the Role of VNUT in Lineage Progression.

Little is known about the intrinsic specification of postnatal cerebellar neural stem cells (NSCs) and to what extent they depend on information from their local niche. Here, we have used an adapted cell preparation of isolated postnatal NSCs and live imaging to demonstrate that cerebellar progenitors maintain their neurogenic nature by displaying hallmarks of NSCs. Furthermore, by using this preparation, all the cell types produced postnatally in the cerebellum, in similar relative proportions to those observed in vivo, can be monitored. The fact that neurogenesis occurs in such organized manner in the absence of signals from the local environment, suggests that cerebellar lineage progression is to an important extent governed by cell-intrinsic or pre-programmed events. Finally, we took advantage of the absence of the niche to assay the influence of the vesicular nucleotide transporter inhibition, which dramatically reduced the number of NSCs in vitro by promoting their progression toward neurogenesis.

Vesicular nucleotide transporter mediates adenosine triphosphate release in compressed human periodontal ligament fibroblast cells and participates in tooth movement-induced nociception in rats.

OBJECTIVE: Pain control is imperative in orthodontic treatment. Adenosine triphosphate (ATP) is a key mediator released from periodontal ligament cells that excites nociceptive nerve endings. Vesicular nucleotide transporter (VNUT), encoded by the Solute carrier family 17 member 9 (SLC17A9) gene, participates in ATP uptake into secretory vesicles; thus, it may mediate tooth movement-induced pain. In the present study, we examined whether VNUT in periodontal ligament cells participates in tooth movement-induced nociception. DESIGN: Expression levels of SLC17A9, connexin 43, and pannexin 1 in human periodontal ligament fibroblasts (HPDLFs) were examined by quantitative reverse transcription-polymerase chain reaction. Mechanical force via centrifugation-induced ATP release was measured using an ATP bioluminescence assay. Inhibitors were used to evaluate the role of ATP transporters. Face-grooming behaviors were assessed as indicators of nociceptive responses after experimental tooth movement in rats, as well as the effects of drugs for the pain-like behavior. RESULTS: After HPDLFs underwent mechanical stimulation by centrifugation, SLC17A9 mRNA expression in the cells was significantly upregulated. Increased ATP release from HPDLFs after mechanical stimulation was suppressed by treatment with clodronic acid, a VNUT inhibitor, at concentrations of 0.1 and 1.0 µM. In rats, face-grooming behaviors (indicators of nociception) were significantly increased on day 1 after experimental tooth movement. Increased face-grooming behaviors were suppressed by systemic administration of clodronic acid (0.1 mg/kg). CONCLUSIONS: These results indicate that release of ATP from periodontal ligament cells via VNUT is important for nociceptive transduction during orthodontic treatment. Thus, VNUT may provide a novel drug target for tooth movement-induced pain.

An insight into the orphan nucleotide sugar transporter SLC35A4.

SLC35A4 has been classified in the SLC35A subfamily based on amino acid sequence homology. Most of the proteins belonging to the SLC35 family act as transporters of nucleotide sugars. In this study, the subcellular localization of endogenous SLC35A4 was determined via immunofluorescence staining, and it was demonstrated that SLC35A4 localizes mainly to the Golgi apparatus. In silico topology prediction suggests that SLC35A4 has an uneven number of transmembrane domains and its N-terminus is directed towards the Golgi lumen. However, an experimental assay refuted this prediction: SLC35A4 has an even number of transmembrane regions with both termini facing the cytosol. In vivo interaction analysis using the FLIM-FRET approach revealed that SLC35A4 neither forms homomers nor associates with other members of the SLC35A subfamily except SLC35A5. Additional assays demonstrated that endogenous SLC35A4 is 10 to 40nm proximal to SLC35A2 and SLC35A3. To determine SLC35A4 function SLC35A4 knock-out cells were generated with the CRISPR-Cas9 approach. Although no significant changes in glycosylation were observed, the introduced mutation influenced the subcellular distribution of the SLC35A2/SLC35A3 complexes. Additional FLIM-FRET experiments revealed that overexpression of SLC35A4-BFP together with SLC35A3 and the SLC35A2-Golgi splice variant negatively affects the interaction between the two latter proteins. The results presented here strongly indicate a modulatory role for SLC35A4 in intracellular trafficking of SLC35A2/SLC35A3 complexes.

Activation of lysosomal P2X4 by ATP transported into lysosomes via VNUT/SLC17A9 using V-ATPase generated voltage gradient as the driving force.

KEY POINTS: SLC17A9 proteins function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation. P2X4 receptors act as lysosomal ion channels activated by luminal ATP. SLC17A9-mediated ATP transport across the lysosomal membrane is suppressed by Bafilomycin A1, the V-ATPase inhibitor. SLC17A9 mainly uses voltage gradient but not pH gradient generated by the V-ATPase as the driving force to transport ATP into the lysosome to activate P2X4. ABSTRACT: The lysosome contains abundant ATP which plays important roles in lysosome functions and in cell signalling. Recently, solute carrier family 17 member 9 (SLC17A9, also known as VNUT for vesicular nucleotide transporter) proteins were suggested to function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation, and P2X4 receptors were suggested to be lysosomal ion channels that are activated by luminal ATP. However, the molecular mechanism of SLC17A9 transporting ATP and the regulatory mechanism of lysosomal P2X4 are largely unknown. In this study, we report that SLC17A9-mediated ATP transport across lysosomal membranes is suppressed by Bafilomycin A1, the V-ATPase inhibitor. By measuring P2X4 activity, which is indicative of ATP transport across lysosomal membranes, we further demonstrated that SLC17A9 mainly uses voltage gradient but not pH gradient as the driving force to transport ATP into lysosomes. This study provides a molecular mechanism for lysosomal ATP transport mediated by SLC17A9. It also suggests a regulatory mechanism of lysosomal P2X4 by SLC17A9.

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells.

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.

Roles of the nucleotide sugar transporters (SLC35 family) in health and disease.

Nucleotide sugars and adenosine 3'-phospho 5'-phosphosulfate (PAPS) are transported from the cytosol to the endoplasmic reticulum (ER) and the Golgi apparatus where they serve as substrates for the glycosylation and sulfation of proteins, lipids and proteoglycans. The translocation is accomplished by the nucleotide sugar transporters (NSTs), a family of highly conserved hydrophobic proteins with multiple transmembrane domains that are part of the solute carrier family 35 (SLC35). NSTs are antiporters responsible not only for transporting nucleotide sugars and PAPS into the Golgi, but also for the transport of the reaction products back to the cytosol. The initial reaction products - the nucleoside diphosphates - must be first converted to nucleoside monophosphates by a group of enzymes called ectonucleoside triphosphate diphosphohydrolases (ENTPDs) before they can exit the Golgi. The transport role of NSTs is essential to glycosylation and development. Mutations in two NST genes, SLC35A1 and SLC35C1, have been related to congenital disorder of glycosylation II (CDG II).

Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria.

Mitochondria transport and utilize iron for the synthesis of haem and Fe-S clusters. Although many proteins are known to be involved in these processes, additional proteins are likely to participate. To test this hypothesis, in the present study we used a genetic screen looking for yeast mutants that are synthetically lethal with the mitochondrial iron carriers Mrs3 and Mrs4. Several genes were identified, including an isolate mutated for Yfh1, the yeast frataxin homologue. All such triple mutants were complemented by increased expression of Rim2, another mitochondrial carrier protein. Rim2 overexpression was able to enhance haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds. Conversely Rim2 depletion impaired haem and Fe-S cluster synthesis in wild-type or Δmrs3/Δmrs4 backgrounds, indicating a unique requirement for this mitochondrial transporter for these processes. Rim2 was previously shown to mediate pyrimidine exchange in and out of vesicles. In the present study we found that isolated mitochondria lacking Rim2 exhibited concordant iron defects and pyrimidine transport defects, although the connection between these two functions is not explained. When organellar membranes were ruptured to bypass iron transport, haem synthesis from added iron and porphyrin was still markedly deficient in Rim2-depleted mitochondrial lysate. The results indicate that Rim2 is a pyrimidine exchanger with an additional unique function in promoting mitochondrial iron utilization.

Connecting the plastid: transporters of the plastid envelope and their role in linking plastidial with cytosolic metabolism.

Plastids have a multitude of functions in eukaryotic cells, ranging from photosynthesis to storage, and a role in essential biosynthetic pathways. All plastids are of either primary or higher-order endosymbiotic origin. That is, either a photosynthetic cyanobacterium was integrated into a mitochondriate eukaryotic host cell (primary endosymbiosis) or a plastid-bearing eukaryotic cell merged with another eukaryotic cell (secondary or higher-order endosymbioses), thereby passing on the plastid between various eukaryotic lineages. For all of these endosymbioses to become functional, it was essential to establish metabolic connections between organelle and host cell. Here, we review the present understanding of metabolite exchange between plastids and the surrounding cytosol in the context of the endosymbiotic origin of plastids in various eukaryotic lineages. We show that only a small number of transporters that can be traced down to the primary endosymbiotic event are conserved between plastids of diverse origins.

Two Golgi-resident 3'-Phosphoadenosine 5'-phosphosulfate transporters play distinct roles in heparan sulfate modifications and embryonic and larval development in Caenorhabditis elegans.

Synthesis of extracellular sulfated molecules requires active 3'-phosphoadenosine 5'-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.

Structural and transcriptional analyses of a purine nucleotide-binding protein from Pyrococcus furiosus: a component of a novel, membrane-bound multiprotein complex unique to this hyperthermophilic archaeon.

The open-reading frame PF0895 in the genome of the hyperthermophilic archaeon, Pyrococcus furiosus, encodes a 206-residue protein (M(R )23,152). The structure of the recombinant protein was solved by single isomorphous replacement with anomalous scattering (SIRAS) using a mercury derivative. It has been refined to 1.70 A with a crystallographic R and R(free )values of 19.7% and 22.3%, respectively. The PF0895 structure is similar to those of the ATP binding cassettes observed in the ABC transporter family. However, bioinformatics and molecular analyses indicate that PF0895 is not part of the expected five-gene operon that encodes a typical prokaryotic solute-binding ABC transporter. Rather, transcriptional profiling data show that PF0895 is part of a novel four-gene operon (PF0895-PF0896-PF0897-PF0897.1) where only PF0895 has homologs in other organisms. Interestingly, from genome analysis, P. furiosus itself contains a second version of this complex, encoded by PF1090-PF1093. From the structural studies we can only conclude that one of the subunits of this novel membrane complex, PF0895, and its homolog PF1090, likely bind a purine nucleotide. PF0895 is therefore predicted to be part of a membrane-bound multiprotein complex unrelated to ABC transporters that is so far unique to P. furiosus. It appears to play a role in the stress response, as its expression is down regulated when the organism is subjected to cold-shock, where cells are transferred from 95 degrees C, near the optimal growth temperature, to 72 degrees C, near the minimal growth temperature. The related PF1090-containing operon is unaffected by cold-shock and is independently regulated.

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human.

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.

The human solute carrier gene SLC35B4 encodes a bifunctional nucleotide sugar transporter with specificity for UDP-xylose and UDP-N-acetylglucosamine.

The transport of nucleotide sugars from the cytoplasm into the Golgi apparatus is mediated by specialized type III proteins, the nucleotide sugar transporters (NSTs). Transport assays carried out in vitro with Golgi vesicles from mammalian cells showed specific uptake for a total of eight nucleotide sugars. When this study was started, NSTs with transport activities for all but two nucleotide sugars (UDP-Xyl and UDP-Glc) had been cloned. Aiming at identifying these elusive NSTs, bioinformatic methods were used to display putative NST sequences in the human genome. Ten open reading frames were identified, cloned, and heterologously expressed in yeast. Transport capabilities for UDP-Glc and UDP-Xyl were determined with Golgi vesicles isolated from transformed cells. Although a potential UDP-Glc transporter could not be identified due to the high endogenous transport background, the measurement of UDP-Xyl transport was possible on a zero background. Vesicles from yeast cells expressing the human gene SLC35B4 showed specific uptake of UDP-Xyl, and subsequent testing of other nucleotide sugars revealed a second activity for UDP-GlcNAc. Expression of the epitope-tagged SLC35B4 in mammalian cells demonstrated strict Golgi localization. Because decarboxylation of UDP-GlcA is known to produce UDP-Xyl directly in the endoplasmic reticulum and Golgi lumen, our data demonstrate that two ways exist to deliver UDP-Xyl to the Golgi apparatus.

The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in DeltapH formation across peroxisomal membranes.

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a DeltapH across the peroxisomal membrane, acidic inside.

Functional differences in nucleoside and nucleobase transporters expressed on the rabbit corneal epithelial cell line (SIRC) and isolated rabbit cornea.

The purpose of this study was to investigate the expression of nucleoside/nucleobase transporters on the Statens Seruminstitut rabbit corneal (SIRC) epithelial cell line and to evaluate SIRC as an in vitro screening tool for delineating the mechanism of corneal permeation of nucleoside analogs. SIRC cells (passages 410-425) were used to study uptake of [3H]thymidine, [3H]adenine, and [3H]ganciclovir. Transport of [3H]adenine and [3H]ganciclovir was studied across isolated rabbit cornea. Uptake and transport studies were performed for 2 minutes and 120 minutes, respectively, at 34 degrees C. Thymidine uptake by SIRC displayed saturable kinetics (K(m) = 595.9 +/- 80.4 microM, and V(max) = 289.5 +/- 17.2 pmol/min/mg protein). Uptake was inhibited by both purine and pyrimidine nucleosides but not by nucleobases. [3H]thymidine uptake was sodium and energy independent but was inhibited by nitrobenzylthioinosine at nanomolar concentrations. Adenine uptake by SIRC consisted of a saturable component (K(m) = 14.4 +/- 2.3 microM, V(max) = 0.4 +/- 0.04 nmol/min/mg protein) and a nonsaturable component. Uptake of adenine was inhibited by purine nucleobases but not by the nucleosides or pyrimidine nucleobases and was independent of sodium, energy, and nitrobenzylthioinosine. [3H]ganciclovir uptake involved a carrier-mediated component and was inhibited by the purine nucleobases but not by the nucleosides or pyrimidine nucleobases. However, transport of [3H]adenine across the isolated rabbit cornea was not inhibited by unlabeled adenine. Further, corneal permeability of ganciclovir across a 100-fold concentration range remained constant, indicating that ganciclovir permeates the cornea primarily by passive diffusion. Nucleoside and nucleobase transporters on rabbit cornea and corneal epithelial cell line, SIRC, are functionally different, undermining the utility of the SIRC cell line as an in vitro screening tool for elucidating the corneal permeation mechanism of nucleoside analogs.