Kikuta S et al. (2012), A novel member of the trehalose transporter fam...

XB-ART-45886

Front Physiol 2012 Jan 01;3:290. doi: 10.3389/fphys.2012.00290.

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A novel member of the trehalose transporter family functions as an h(+)-dependent trehalose transporter in the reabsorption of trehalose in malpighian tubules.

Kikuta S , Hagiwara-Komoda Y , Noda H , Kikawada T .

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In insects, Malpighian tubules are functionally analogous to mammalian kidneys in that they not only are essential to excrete waste molecules into the lumen but also are responsible for the reabsorption of indispensable molecules, such as sugars, from the lumen to the principal cells. Among sugars, the disaccharide trehalose is highly important to insects because it is the main hemolymph sugar to serve as a source of energy and carbon. The trehalose transporter TRET1 participates in the transfer of newly synthesized trehalose from the fat body across the cellular membrane into the hemolymph. Although transport proteins must play a pivotal role in the reabsorption of trehalose in Malpighian tubules, the molecular context underlying this process remains obscure. Previously, we identified a Tret1 homolog (Nlst8) that is expressed principally in the Malpighian tubules of the brown planthopper (BPH). Here, we used the Xenopus oocyte expression system to show that NlST8 exerts trehalose transport activity that is elevated under low pH conditions. These functional assays indicate that Nlst8 encodes a proton-dependent trehalose transporter (H-TRET1). To examine the involvement of Nlst8 in trehalose reabsorption, we analyzed the sugar composition of honeydew by using BPH with RNAi gene silencing. Trehalose was detected in the honeydew as waste excreted from Nlst8-dsRNA-injected BPH under hyperglycemic conditions. However, trehalose was not expelled from GFP-dsRNA-injected BPH even under hyperglycemic conditions. We conclude that NlST8 could participate in trehalose reabsorption driven by a H(+) gradient from the lumen to the principal cells of the Malpighian tubules.

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Species referenced: Xenopus
Genes referenced: agxt mtnr1al slc45a3 tbx2 tirap trhd

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	Figure 1. Functional analyses of NlST8-expressing Xenopus oocyte. Localization of NlST8 in the membrane of the Xenopus oocyte as detected by use of an AcGFP1 fusion protein. (A) NlST8::AcGFP1 fluorescence was observed in Xenopus oocytes. (B) AcGFP1 cRNA injection as a control. (C) Sham as a negative control. Scale bar, 20âÎ¼m. (D) Sugar uptake analyses of NlST8 by using HPLC. Transporters were expressed in the cellular membrane of Xenopus oocytes by injecting the cRNA of Nlst8. 2-DOG, 2-deoxy-glucose; Myo, myo-inositol; Suc, sucrose; Mal, maltose; Tre, Trehalose. Sham is a negative control. Sugars were used at a concentration of 105âmM in MBS buffer. Ten oocytes were analyzed in each assay. Error bars represent the standard error (nâ=â3). Statistical significance was determined by using Studentâs t-test in each assay. ânsâ indicates no significant difference; asterisks indicate a significant difference (****Pâ<â0.0001). Myo-inositol: Pâ=â0.2626, sucrose: Pâ=â0.8057, maltose: Pâ=â0.1241, and trehalose: Pâ<â0.0001.
	Figure 2. Trehalose uptake via NlST8 is driven at low pH. Ten oocytes were analyzed in each assay; error bars represent the standard error (nâ=â3). The trehalose concentration used was 105âmM. (A) Trehalose uptake was estimated under Na+-free conditions. Statistical significance was determined by using Studentâs t-test in the assays; ânsâ indicates no significant difference (Pâ=â0.7686). (B) Trehalose uptake was examined under various pH conditions. Statistical analyses were performed by using one-way ANOVA before Tukeyâs multiple comparison tests. Columns labeled with the same letters indicate no significant difference (Pâ>â0.05). (C) Trehalose uptake in the presence of the protonophore CCCP (50âÎ¼M). Gray bars show trehalose uptake at pH 5 in 105âmM trehalose solution, and white bars show trehalose uptake at pH 7.8.
	Figure 3. Analyses of the kinetics of NlST8 for trehalose. Ten oocytes were analyzed in each assay; error bars represent the standard error (nâ=â3). (A) Oocytes expressing NlST8 were incubated with various trehalose concentrations for 3âh at pH 7.8. (B) Oocytes expressing NlST8 were incubated for 1âh with various trehalose concentrations at pH 5. Data were fitted to the MichaelisâMenten equation.
	Figure 4. Reabsorption of trehalose in BPH. (A) Experimental scheme of hemolymph and honeydew analysis. RNAi techniques and quantification of sugars in hemolymph and honeydew are described in the Appendix. Briefly, BPH female adults were injected with dsRNA solution in the segment between the thorax and abdomen and incubated for 24âh to suppress gene expression. Each EGFP1 or Nlst8 RNAi BPH was then injected with 50ânl of water or trehalose in the same segment and incubated for 24âh in a Parafilm sachet. Finally, sugars in BPH hemolymph or in honeydew were analyzed by using HPLC. Sugar contents are represented as percentages (mg/100âÎ¼l). (B) Effect of RNAi on the gene expression levels of Nlst8 at 48âh after injection. EGFP1-dsRNA served as a negative control. Error bars represent the standard error (nâ=â3). (C) Hemolymph sugar contents following injection of water or 1âM trehalose into EGFP1 (upper) or Nlst8 (lower) RNAi BPH. Sugar contents were analyzed at 30âmin after injection (left), and at 24âh after injection (right). (D) Quantifications of trehalose in BPH honeydew. Statistical analyses were performed by using the KruskalâWallis test before Dunnâs multiple comparison tests. The asterisk indicates a significant difference (Pâ=â0.0023).
	Figure 5. Schematic model of trehalose reabsorption. Principal cells mediate proton transfer, driven by a V-type H+ ATPase, with H-TRET1 (NlST8) putatively expressed in the apical membrane. Trehalose in the hemolymph that leaks via the paracellular transport pathway through the septate junction would be reabsorbed by H-TRET1 driven by the H+ electrochemical membrane potentials from the tubule lumen to the principal cells. This trehalose would then be hydrolyzed into two glucose molecules by trehalase. Glucose would be utilized as an energy source for the proton transfer driven by the V-type H+ ATPase.
	Figure A1. Phylogenetic analysis of the deduced amino acid sequences of putative trehalose transporters. A rooted tree was constructed with the neighbor-joining method using MEGA ver. 5 (Tamura et al., 2011), and the tree was tested with a bootstrap analysis of 1,000 replications. The insect trehalose transporters were: NlST8 from N. lugens (NCBI accession number: AB550001), AgTRET1 from A. gambiae (AB369548), PvTRET1 from P. vanderplanki (AB272983), and DmTRET 1-1B from D. melanogaster (AB369553). The glucose transporter NlST1 of N. lugens (AB549994) was applied as an out-group of the trehalose transporter family. SCRT (CG4484), derived from D. melangaster, is a highly similar protein that belongs to the mammalian solute carrier family 45 (SLC45) and is an H+-sucrose co-transporter. The other amino acid sequences, AGT1 of S. cerevisiae, SpSUT1 of Schizosaccharomyces pombe (NP_594387.1), human SLC45A3 (NP_149093), and human hGLUT1 (NP_006507.2) were obtained from the NCBI public database.
	Figure A2. Trehalose uptake into Xenopus oocytes via PvTRET1 at various pH values. Ten oocytes were analyzed in each assay; error bars represent the standard error (nâ=â3). The PvTRET1-expressing oocytes were incubated in 105âmM trehalose solutions. Statistical significance was determined by using one-way ANOVA. ânsâ indicates no significant difference (Pâ=â0.845).
	Figure A3. Trehalose transport via NlST8 is proton-dependent. Trehalose uptake into oocytes expressing NlST8 was examined at pH 5 with increasing concentrations of the protonophore CCCP (0, 10, and 50âÎ¼M). Trehalose uptake at pH 7.8 is shown as a negative control. Ten oocytes were analyzed in each assay; error bars represent the standard error (nâ=â3). The trehalose concentration in the buffer was 105âmM.
	Figure A4. Detection of sugars in BPH honeydew. (A) HPLC chromatogram representing the sugars present in BPH honeydew. White arrowhead shows sorbitol as an internal standard control; black arrowheads show the sugars detected in honeydew. Frc: fructose, Sor: sorbitol, Glc: glucose, Suc: sucrose, Mel: melezitose. (B) Retention time of trehalose and sorbitol. (C) Trehalose detection in BPH honeydew. Black arrowhead represents the trehalose (Tre) peak.
	Figure A5. Nlst8 gene expression by using realtime RT-PCR (modified from Figure 1 in Kikuta et al., 2010). Tissues were independently prepared three times. RP-L4 was used for normalization. Tissue abbreviations: WB, whole-body; HD, head; TH, thorax; AB, abdomen; MG, midgut; OV, ovary; TS, testis; SG, salivary glands; MT, Malpighian tubules, and FB, fat body. Error bars represent the standard deviation.
	Figure A6. Time course of trehalose uptake in NlST8-expressing Xenopus oocytes. Ten oocytes were analyzed in each assay; error bars represent the standard error (nâ=â3). Sham-treated oocytes served as a control. The trehalose concentration in the buffer was 105âmM.

References [+] :

Arrese, Insect fat body: energy, metabolism, and regulation. 2010, Pubmed