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Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance.

Söderberg JA, Birse RT, Nässel DR - PLoS ONE (2011)

Bottom Line: Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype.Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation.Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Stockholm University, Stockholm, Sweden.

ABSTRACT
The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7) are known, some of which are produced in the brain, others in fat body or intestine. Here we show that DILP5 is expressed in principal cells of the renal tubules of Drosophila and affects survival at stress. Renal (Malpighian) tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. We investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK) and its receptor DTKR during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone.

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Expression of DILP5, tachykinin receptor, and insulin receptor in renal tubules.A-C. DILP5 immunolabeling in principal cells of adult tubules (A, B) and in tubules of third instar larva (C). A surface view is seen in D and an optical section with tubule lumen in E and F. D, E. Detection of DILP2 immunolabeling in the principal cells (identified by c42-Gal4-GFP in D). The antibody raised against DILP2 cross-reacts with DILP5 present in the principal cells. F. RT-PCR shows that Dilp5 transcript is present both in renal tubules (RT) and brains of larvae (L3) and adults. Predicted product size is 211 bp for Dilp5. The bands were cut out and sequenced: the upper band in each lane represents Dilp5 and the lower bands in RT are degenerate Dilp-5 sequences). G. RT-PCR identifying Dilp2 transcript (predicted 183 bp) in brain of larvae and adults, but not in the renal tubules. The upper band in each lane is Rp49 transcript as loading control. Also the other Dilp transcripts were analyzed in the tubules; only Dilp5 was detected (See S. Fig. 2). H. C324-Gal4 driven GFP expression in the principal cells. I. C724-Gal4 driven GFP expression is seen in the stellate cells. K. Immunofluorescent detection of DTKR in the principal cells in adult renal tubules. Note that the intercalating stellate cells do not express the receptor. L, M. Antiserum to the insulin receptor dInR labels the principal cells. Note that stellate cells are not labeled (arrow in L).
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pone-0019866-g001: Expression of DILP5, tachykinin receptor, and insulin receptor in renal tubules.A-C. DILP5 immunolabeling in principal cells of adult tubules (A, B) and in tubules of third instar larva (C). A surface view is seen in D and an optical section with tubule lumen in E and F. D, E. Detection of DILP2 immunolabeling in the principal cells (identified by c42-Gal4-GFP in D). The antibody raised against DILP2 cross-reacts with DILP5 present in the principal cells. F. RT-PCR shows that Dilp5 transcript is present both in renal tubules (RT) and brains of larvae (L3) and adults. Predicted product size is 211 bp for Dilp5. The bands were cut out and sequenced: the upper band in each lane represents Dilp5 and the lower bands in RT are degenerate Dilp-5 sequences). G. RT-PCR identifying Dilp2 transcript (predicted 183 bp) in brain of larvae and adults, but not in the renal tubules. The upper band in each lane is Rp49 transcript as loading control. Also the other Dilp transcripts were analyzed in the tubules; only Dilp5 was detected (See S. Fig. 2). H. C324-Gal4 driven GFP expression in the principal cells. I. C724-Gal4 driven GFP expression is seen in the stellate cells. K. Immunofluorescent detection of DTKR in the principal cells in adult renal tubules. Note that the intercalating stellate cells do not express the receptor. L, M. Antiserum to the insulin receptor dInR labels the principal cells. Note that stellate cells are not labeled (arrow in L).

Mentions: Gene microarray data has revealed enrichment of mRNA of DILP5, one of the seven known DILPs, in larval renal tubules of larval Drosophila (see FlyAtlas http://flyatlas.org/[34]). Encouraged by this we developed an antiserum to the C-chain of DILP5 and show here immunolabeling of principal cells of the renal tubules in both adults and third instar larvae (Fig. 1A–C). Also an antiserum to the A-chain of DILP2 [35], that cross reacts with DILP5 due to sequence similarities, labeled these cells (Fig. 1D,E). As a control we showed that over-expression of DILP2, using the Gal4 line C324 specific for principal cells (Fig. 1H), crossed with UAS-Dilp2, resulted in strongly increased immunolabeling of principal cells with the DILP2 antiserum (Fig. S1A,C). This confirmed both that the antiserum recognizes ectopic DILP2 and that the principal cells can produce DILPs. We also used targeted RNA interference (RNAi) with the transgene C324-Gal4/UAS-Dilp5-RNAi to knock down DILP5 in principal cells and found that immunolabeling with either of the DILP2 and DILP5 antisera was strongly reduced in the tubules (Fig. S1B, C). Therefore, both the general DILP2 antiserum and the specific DILP5 antiserum recognize DILP5, and furthermore the Dilp5-RNAi efficiently reduces the peptide level in the principal cells. Next, we confirmed the presence of RNA encoding Dilp5 in dissected renal tubules of larvae and adults by RT-PCR (Fig. 1F). As a comparison Dilp2 transcript was found in the brain, but not in renal tubules (Fig. 1G). We also examined whether transcripts of the other Dilps are present in renal tubules and found that only Dilp5 is present (Fig. S2)


Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance.

Söderberg JA, Birse RT, Nässel DR - PLoS ONE (2011)

Expression of DILP5, tachykinin receptor, and insulin receptor in renal tubules.A-C. DILP5 immunolabeling in principal cells of adult tubules (A, B) and in tubules of third instar larva (C). A surface view is seen in D and an optical section with tubule lumen in E and F. D, E. Detection of DILP2 immunolabeling in the principal cells (identified by c42-Gal4-GFP in D). The antibody raised against DILP2 cross-reacts with DILP5 present in the principal cells. F. RT-PCR shows that Dilp5 transcript is present both in renal tubules (RT) and brains of larvae (L3) and adults. Predicted product size is 211 bp for Dilp5. The bands were cut out and sequenced: the upper band in each lane represents Dilp5 and the lower bands in RT are degenerate Dilp-5 sequences). G. RT-PCR identifying Dilp2 transcript (predicted 183 bp) in brain of larvae and adults, but not in the renal tubules. The upper band in each lane is Rp49 transcript as loading control. Also the other Dilp transcripts were analyzed in the tubules; only Dilp5 was detected (See S. Fig. 2). H. C324-Gal4 driven GFP expression in the principal cells. I. C724-Gal4 driven GFP expression is seen in the stellate cells. K. Immunofluorescent detection of DTKR in the principal cells in adult renal tubules. Note that the intercalating stellate cells do not express the receptor. L, M. Antiserum to the insulin receptor dInR labels the principal cells. Note that stellate cells are not labeled (arrow in L).
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pone-0019866-g001: Expression of DILP5, tachykinin receptor, and insulin receptor in renal tubules.A-C. DILP5 immunolabeling in principal cells of adult tubules (A, B) and in tubules of third instar larva (C). A surface view is seen in D and an optical section with tubule lumen in E and F. D, E. Detection of DILP2 immunolabeling in the principal cells (identified by c42-Gal4-GFP in D). The antibody raised against DILP2 cross-reacts with DILP5 present in the principal cells. F. RT-PCR shows that Dilp5 transcript is present both in renal tubules (RT) and brains of larvae (L3) and adults. Predicted product size is 211 bp for Dilp5. The bands were cut out and sequenced: the upper band in each lane represents Dilp5 and the lower bands in RT are degenerate Dilp-5 sequences). G. RT-PCR identifying Dilp2 transcript (predicted 183 bp) in brain of larvae and adults, but not in the renal tubules. The upper band in each lane is Rp49 transcript as loading control. Also the other Dilp transcripts were analyzed in the tubules; only Dilp5 was detected (See S. Fig. 2). H. C324-Gal4 driven GFP expression in the principal cells. I. C724-Gal4 driven GFP expression is seen in the stellate cells. K. Immunofluorescent detection of DTKR in the principal cells in adult renal tubules. Note that the intercalating stellate cells do not express the receptor. L, M. Antiserum to the insulin receptor dInR labels the principal cells. Note that stellate cells are not labeled (arrow in L).
Mentions: Gene microarray data has revealed enrichment of mRNA of DILP5, one of the seven known DILPs, in larval renal tubules of larval Drosophila (see FlyAtlas http://flyatlas.org/[34]). Encouraged by this we developed an antiserum to the C-chain of DILP5 and show here immunolabeling of principal cells of the renal tubules in both adults and third instar larvae (Fig. 1A–C). Also an antiserum to the A-chain of DILP2 [35], that cross reacts with DILP5 due to sequence similarities, labeled these cells (Fig. 1D,E). As a control we showed that over-expression of DILP2, using the Gal4 line C324 specific for principal cells (Fig. 1H), crossed with UAS-Dilp2, resulted in strongly increased immunolabeling of principal cells with the DILP2 antiserum (Fig. S1A,C). This confirmed both that the antiserum recognizes ectopic DILP2 and that the principal cells can produce DILPs. We also used targeted RNA interference (RNAi) with the transgene C324-Gal4/UAS-Dilp5-RNAi to knock down DILP5 in principal cells and found that immunolabeling with either of the DILP2 and DILP5 antisera was strongly reduced in the tubules (Fig. S1B, C). Therefore, both the general DILP2 antiserum and the specific DILP5 antiserum recognize DILP5, and furthermore the Dilp5-RNAi efficiently reduces the peptide level in the principal cells. Next, we confirmed the presence of RNA encoding Dilp5 in dissected renal tubules of larvae and adults by RT-PCR (Fig. 1F). As a comparison Dilp2 transcript was found in the brain, but not in renal tubules (Fig. 1G). We also examined whether transcripts of the other Dilps are present in renal tubules and found that only Dilp5 is present (Fig. S2)

Bottom Line: Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype.Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation.Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, Stockholm University, Stockholm, Sweden.

ABSTRACT
The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7) are known, some of which are produced in the brain, others in fat body or intestine. Here we show that DILP5 is expressed in principal cells of the renal tubules of Drosophila and affects survival at stress. Renal (Malpighian) tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. We investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK) and its receptor DTKR during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. Our findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone.

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