npfr1 nat cell biol

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Drosophila short neuropeptide F signalling regulates growth by erK-mediated insulin signallingKyu-Sun Lee1*, O-Yu Kwon2*, Joon H. Lee3*, Kisang Kwon2, Kyung-Jin Min4†, Sun-Ah Jung3, Ae-Kyeong Kim1, Kwan-Hee You5, Marc Tatar4 & Kweon Yu1,6

Insulin and insulin growth factor have central roles in growth1,2, metabolism3 and ageing4,5 of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain3,6. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.

Neuropeptides regulate a wide range of animal behaviours related to nutrition7–9. In particular, mammalian NPY produced in the hypotha-lamus of the brain controls food consumption. NPY injection in the hypothalamus of rats produces hyperphagia and obese phenotypes8,9. The Drosophila orthologue of NPY is sNPF. This peptide is expressed in the nervous system and it regulates food intake and body size; over-expression of sNPF produces bigger and heavier flies10. Likewise, the G-protein-coupled receptor of sNPF (sNPFR1) is expressed in neurons and shows significant similarity with vertebrate NPY receptors11. In both mammals, however, little is known about how NPY and sNPF systemi-cally modulate growth, metabolism and lifespan. Here we show that

these neuropeptides control expression of insulin-like peptides and subsequently affect insulin signalling in target tissues.

Initially we characterized the effects of sNPF and sNPFR1 on body size by measuring the length of flies from head to abdomen. The body size of sNPF hypomorphic Drosophila mutants (sNPFc00448) was 23% of that of the wild-type, whereas overexpressing two copies of the sNPF in the sensory neurons and sensory structures of the nervous systems by MJ94-Gal412, 13 (MJ94>2XsNPF) increased body size by 24% (Fig. 1a, b, asterisks). Similar changes were seen in the overall size of adult wings, which resulted from changes in both cell size and number (Fig. S1a–c). Effects on body size were associated with sNPF expression levels (Fig. S2a): relative to wild type, sNPF levels were 3.5-fold higher in MJ94>2XsNPF and less than half of the wild type in sNPFc00448. In contrast to the effect of sNPF on body size, there was little effect on size from repression or overexpression of the sNPF receptor in MJ94-expressing cells (Fig. 1b).

Drosophila insulin-like peptides (Dilps) modulate growth and adult size; therefore, we tested whether sNPF has a role in insulin-producing neurons. For positive controls, we overexpressed Dilp2 in insulin-producing cells (IPCs) through Dilp2–Gal4, which increased body size, and we ablated the IPCs by expression of Dilp2>reaper to decrease body size (Fig. 1c). To investigate sNPF signalling, we overexpressed sNPFR1 in the IPCs and observed a 10% increase in body size. Conversely, expression of the sNPFR1 dominant-negative mutant (Dilp2>sNPFR1-DN) reduced body size by 14% (Fig. 1c, asterisks). Manipulation of the sNPF ligand with IPCs expressing Dilp2–Gal4, however, did not affect body size: flies overexpressing sNPF (Dilp2>2XsNPF) or in which sNPF was silenced RNAi (Dilp2>sNPF-Ri) were of similar size to the wild type (Fig. 1c). Taken together, these results suggest that sNPF peptide may be secreted from MJ94-expressing sensory neurons and activate sNPFR1 of Dilp2-expressing IPCs.

To assess this model we visualized the sNPF ligand and sNPFR1 receptor in the larval brain. Seven IPCs were detected in each brain hemisphere using the marker Dilp2–Gal4>nGFP (Fig. 1d, arrows). Neurons containing sNPF peptide in the axon terminal and cell body

1Centre for Regenerative Medicine, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-333, Korea. 2Department of Anatomy, College of Medicine, 5School of Bioscience and Biotechnology, Chungnam National University, Daejeon, Korea. 3Myung-Gok Eye Research Institute, Kim’s Eye Hospital, College of Medicine, Konyang University, Chungnam, Korea. 4Department of Ecology and Evolutionary Biology, Brown University, Providence, RI 02912, USA.*These authors contributed equally to this work.†Present address: Department of Biological Sciences, University of Alaska at Anchorage , Anchorage, AK 99508, USA.6Correspondence should be addressed to K.Y. ([email protected])

Received 7 December 2007; accepted 6 February 2008: published online 16 March 2008; DOI: 10.1038/ncb1710

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(sNPFnergic neurons) were stained adjacent to these IPCs (Fig. 1d). As expected, sNPFR1 receptors were localized in the plasma membrane of IPCs marked with Dilp2>DsRed (Fig. 1e, arrows; Fig. S3a–c). sNPFR1

was also localized in the neurons of the larval brain hemispheres, sub-oesophagus ganglion, ventral abdominal neurons and descending axons in the ventral ganglion (Fig. S3d).

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Figure 1 Body sizes of sNPF and sNPFR1 mutants, genetic interactions between sNPFR and Dilps and localization of sNPF and sNPFR1 proteins in the larval brain. (a) Body sizes of the hypomorphic sNPFc00448 mutant, wild type and the sNPF overexpression mutant produced by overexpressing two copies of sNPF in the nervous system (MJ94>2XsNPF). The scale bar is 1 mm. (b) The sNPFc00448 hypomorphic mutant was 23% smaller than the wild type, and the MJ94>2XsNPF overexpression mutant was 24% bigger than the control flies (asterisks on the figure). The ANOVA test was used to compare the genotypes WT and MJ94>2XsNPF, P=5.3×10-14; WT and sNPFc00448, P = 1.6×10–17. (c) IPCs overexpressing sNPF receptor (Dilp2>sNPFR1) were 10% bigger than wild type, whereas IPCs with repression of sNPFR1 (Dilp2>sNPFR1-DN) were 14% smaller than the control flies (asterisks). Overexpressing Dilp2 in IPCs (Dilp2 >Dilp2) produced 25% bigger and ablating IPCs by introducing Dilp2>reaper produced flies that were 34%

smaller than the control flies. In common with the reduced body size produced by the inhibition of Dilp1 and Dilp2 in IPCs (Dilp2>Dilp1-Ri and Dilp2>Dilp2-Ri), inhibition of Dilp1 and Dilp2 with sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+Dilp1-Ri and Dilp2> sNPFR1+Dilp2-Ri) also generated reduced body size by 8% and 13%, respectively (double asterisks). In the ANOVA test: P = 0.0005 (Dilp2>sNPFR1); P = 0.0002 (Dilp2>sNPFR1-DN); P = 0.02 (Dilp2> sNPFR1+DiIp1-Ri); P = 0.001 (Dilp2>sNPFR1+DiIp2-Ri). The error bars represent s.e.m. and n = 30 in all genotypes. (d) Seven Dilp2-producing IPCs of each brain hemisphere were marked by Dilp2>nGFP (green, arrows) and the sNPF antibody detected sNPF peptides in the axon terminals and cell bodies of sNPFnergic neurons (red). sNPFnergic neurons were adjacent to IPCs. (e) sNPFR1 antibody staining (green) and Dilp2-producing IPCs marked by Dilp2>DsRed (red) show the localization of sNPFR in the plasma membrane of IPCs (yellow, arrows). The scale bars for d, e are 100 µm.

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To study genetic interactions between sNPFR1 and Dilps in IPCs, we generated Dilp1 and Dilp2 interference mutants in the sNPFR1 over-expression background. In contrast to the 10% body size increase by sNPFR1 overexpression in IPCs (Dilp2>sNPFR1), inhibition of Dilp1 and Dilp2 in IPCs (Dilp2>Dilp1-Ri and Dilp2>Dilp2-Ri) generated reduced

body size by 10% and 15%, respectively. Inhibition of Dilp1 and Dilp2 with sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+Dilp1-Ri and Dilp2> sNPFR1+Dilp2-Ri) also generated a reduction in body size of 8% and 13% (Fig. 1c, double asterisks), indicating that Dilp1 and Dilp2 are downstream genes of sNPFR1 in IPCs for regulating body size.

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Figure 2 sNPF and sNPFR1 regulate expression of Dilps and ERK activation. (a) Dilp2 mRNA expression in the IPCs of the wild-type larval brain (arrowheads). (b) Neuronal overexpression of sNPF (MJ94>2XsNPF) markedly increased Dilp2 expression in IPCs (arrowheads) and produced novel Dilp2 expression (arrows). (c) Reduction of sNPF by MJ94>sNPF-Ri inhibited expression of Dilp2 (arrowheads). Scale bars for a, b, c are 100 µm. (d) Expression of Dilp1 and Dilp2 was positively regulated by sNPF overexpression and reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448). (e) Expression of Dilp1 and Dilp2 was increased with overexpression of the receptor in IPCs (Dilp2>sNPFR1) more than fourfold and decreased with IPCs inhibition of the receptor gene (Dilp2>sNPFR1-DN)

by half. (f) sNPF overexpression with MJ94-Gal4 increased phospho-activated pERK relative to basal ERK1/2. Expression of the receptor sNPFR1 in IPCs also increased pERK. β-actin was used as the control. Whole gel scans are shown in Fig. S6a. (g) Expression of a constitutively active ERK in IPCs (Dilp2>rolledSEM) increased expression of Dilp1 and Dilp2, and both transcripts were repressed by the expression of an ERK inhibitory phosphatase in the IPCs (Dilp2>DMKP-3). The inhibition of ERK with sNPFR1 overexpressed in IPCs (Dilp2>sNPFR1+DMKP-3) repressed expression of Dilp1 and Dilp2 compared with that by sNPFR1 overexpression in IPCs (Dilp2>sNPFR1) (compare Fig. 2g with Fig. 2e). The error bars represent s.e.m. from three independent experiments.



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To test whether sNPF regulates Dilp expression in larval IPCs, we assessed expression of Dilp1, 2, 3 and 5 in sNPF mutants. Neuronal over-expression of sNPF (MJ94>2XsNPF) markedly increased expression of Dilp2 in IPCs; it also produced novel Dilp2 expression outside of these cells (compare Fig. 2b with Fig. 2a, arrowheads and arrows). As expected, reduction of sNPF by MJ94>sNPF-Ri inhibited expression of Dilp2 (Fig. 2c, arrowheads). In common with Dilp2, the expression of Dilp1 was posi-tively regulated by sNPF overexpression and reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448) (Fig. 2d). Consistent with our model, expression of Dilp1 and Dilp2 was increased more than fourfold with overexpression of the receptor in IPCs (Dilp2>sNPFR1) and decreased by half with inhibition of the receptor gene in IPCs (Dilp2>sNPFR1-DN) (Fig. 2e). Larval IPCs also express Dilp3 and Dilp5 (refs 3, 6). We found

that expression of Dilp3 was reduced by sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448) but expression of Dilp5 was not regulated by any sNPF mutants (Fig. S2b, c). There are few functions known to distinguish these various insulin-like peptides. Nutrition-dependent growth regulation is associated with expression of Dilp3 and Dilp5, but not with that of Dilp2 (refs 2, 14). Recent reports show that Dilp2 is reduced in long-lived flies expressing dFOXO or Jun-N-terminal kinase (JNK)15,16, whereas Dilp5 is uniquely upregulated upon dietary restriction that increases lifespan (data not shown).

NPY activates ERK signalling to stimulate growth of mouse pancre-atic β-cells17. To investigate how Drosophila sNPF regulates Dilp expres-sion, we measured the activation of Drosophila MAP kinase signalling, which includes the action of ERK (encoded by Rolled) and JNK. sNPF

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Figure 3 sNPF and NPY peptide regulate ERK-mediated insulin expression in the Drosophila CNS BG2-c6 cells and rat insulinoma INS-1 cells respectively. (a) In the sNPF-peptide-treated Drosophila BG2-c6 cells, Dilp1 and Dilp2 were induced within 15 min, and elevated expression persisted for 1 h. (b) ERK was activated at 15 min. Whole gel scans are shown in Fig. S6c. (c) When cells were treated with sNPF peptide and ERK-specific kinase

MEK inhibitor PD98059, expression of Dilp1 and Dilp2 was not significantly increased. (d) In the human NPY-peptide-treated rat INS-1 cells, expression of insulin1 and insulin2 was activated within 15 min. (e) ERK was activated at 15 min. Whole gel scans are shown in Fig. S6d. (f) Treatment with NPY and MEK inhibitor PD98059 abolished the induction of insulin1 and insulin2. The error bars represent s.e.m. from three independent experiments.



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overexpression with MJ94-Gal4 increased phospho-activated pERK relative to basal ERK1/2. Expression of the receptor protein sNPFR1 in IPCs also increased pERK (Fig. 2f, Fig. S4). There were no detectable

changes in phospho-activated pJNK in these sNPF and sNPFR1 mutants (data not shown). Next, we tested whether ERK activation in IPCs was sufficient to induce Dilp expression. Expression of a constitutively active

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Figure 4 sNPF regulates insulin receptor/dFOXO signalling in fat body cells. (a, d, h, l, p) Fat body cells in animals with neuronal overexpression of sNPF (MJ94>2XsNP) were 42% larger than controls, whereas inhibition of sNPF by MJ94>sNPF-Ri and sNPFc00448 reduced cell size by 38% and 51%, respectively. The ANOVA test was used to compare the genotypes: WT and MJ94>2XsNPF, P=1.7×10–9; WT and MJ94>sNPF-Ri, P=3.0×10-

14, and WT and sNPFc00448, P=1.0×10–6. The error bars represent s.e.m. and n=20 in all genotypes. (b) Overexpression of sNPF (MJ94>sNPF and MJ94>2XsNPF) leads to phosphorylation and activation of Akt in the fat body, whereas the opposite effects were seen with neuronal inhibition

of sNPF (MJ94>sNPF-Ri and sNPFc00448). Whole gel scans are shown in Supplementary Fig. S6b. (c) Expression of d4E-BP was elevated in sNPF hypomorphs (MJ94>sNPF-Ri and sNPFc00448) and reduced upon sNPF overexpression (MJ94>2XsNPF). The error bars represent s.e.m. from three independent experiments. (e–g) In the wild-type fat body cells, dFOXO was localized in the cytoplasm and nucleus. (i–k) Neuronal induction of sNFP (MJ94>2XsNPF) increased the cytoplasmic localization of dFOXO. (m–o, q–s) Reduction of sNPF (MJ94>sNPF-Ri and sNPFc00448) yielded fat body cells with dFOXO predominantly localized in the nucleus. Scale bars are 100 µm.



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ERK in IPCs (Dilp2>rolledSEM) increased expression of Dilp1 and Dilp2 more than threefold, and both transcripts were repressed less than half by the expression of an ERK inhibitory phosphatase DMKP-3 (ref. 18) in IPCs (Dilp2>DMKP-3) (Fig. 2g). In addition, the inhibition of ERK with the sNPFR1 overexpression in IPCs (Dilp2>sNPFR1+DMKP-3) also repressed expression of Dilp1 and Dilp2 compared with that of sNPFR1 overexpression in IPCs (Dilp2>sNPFR1) (compare Fig. 2g with Fig. 2e). These results indicate that sNPF and sNPFR1 signalling regulate ERK activation in IPCs, which in turn modulates expression of Dilp1 and Dilp2.

To further examine the effect of sNPF on Dilp, we treated Drosophila CNS-derived neural BG2-c6 cells19, which endogenously express sNPFR1 (Fig. S5a, b) with a synthetic sNPF peptide. Dilp1 and Dilp2 were induced within 15 min, and the elevated transcript persisted for 1 h (Fig. 3a). Concomitant with this gene expression, sNPF-treated cells activated ERK (Fig. 3b). Importantly, sNPF did not induce Dilp expression significantly when cells were treated with ERK-specific kinase MEK inhibitor PD98059 (Fig. 3c). To compare the functional conservation of sNPF and NPY in the regulation of insulin expression, we conducted similar tests with rat insulinoma INS-1 cells20, which express NPY receptors NPYR1 and

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Figure 5 sNPF regulates the level of haemolymph carbohydrates and lifespan. (a) Trehalose and glucose carbohydrates were reduced upon sNPF overexpression (WT and MJ94>2XsNPF, P=4.6×10–6), and both were elevated in sNPF hypomorphs (WT and MJ94>sNPF-Ri, P=2.3×10–5; WT and sNPFc00448, P=2.7×10–7). The carbohydrate level in Dilp2>reaper, a Drosophila diabetic model, was 48% higher than in the wild-type control (P=8.7×10–11). The error bars represent s.e.m from 10 independent experiments. (b, c) sNPF overexpression in neurons (MJ94>2xsNPF) did

not affect lifespan (Log rank test, UAS-2XsNPF versus MJ94>2xsNPF, χ2=0.073, P=0.786) but sNPF inhibition (MJ94>sNPF-Ri) increased median lifespan by 16–21% (Log rank test, MJ94-gal4 versus MJ94>sNPF-Ri, χ2=18.59, P<0.0001; UAS–sNPF-Ri vs MJ94>sNPF-Ri, χ2=119.26, P<0.0001). (d) Our model for this work. sNPF and sNPFR1 signalling turns on Dilp1 and Dilp2 transcripts through ERK activation in IPCs, and the secreted Dilps affect InR/dFOXO signalling in target tissue, which modulate growth, metabolism and lifespan.



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NPRY2 (Fig. S5c). When treated with the human NPY peptide, expression of insulin1 and insulin2 and ERK was activated within 15 min (Fig. 3d, e). Furthermore, treatment with the MEK inhibitor PD98059 and NPY abolished the induction of insulin1 and insulin2 (Fig. 3f). Together, these findings suggest that the regulation of insulin expression by sNPF or NPY through ERK is evolutionarily conserved in Drosophila and mammals.

To verify that sNPF induction of Dilp expression has a physiological consequence, we analysed insulin signals at a target tissue, the Drosophila fat body1–3. Fat body cells in flies with neuronal overexpression of sNPF (MJ94>2XsNP) were 42% larger than in the control, whereas inhibition of sNPF by MJ94>sNPF-Ri and sNPFc00448 reduced cell size by 38% and 51% respectively (Fig. 4a, d, h, l, p). These differences in size correspond to changes in insulin signal transduction within the cells. Overexpression of sNPF (MJ94>sNPF and MJ94>2XsNPF) leads to phosphorylation and activation of Akt in the fat body, whereas the opposite effect was seen with neuronal inhibition of sNPF (MJ94>sNPF-Ri and sNPFc00448) (Fig. 4b). Activated Akt represses the transcription factor dFOXO by phosphorylation and subsequent cytoplasmic localization. In wild-type flies, dFOXO localized equally in the cytoplasm and nucleus (Fig. 4e–g). As predicted, neuronal induction of sNFP (MJ94>2XsNPF) increased the cytoplasmic localization of dFOXO (Fig. 4i–k), whereas inhibition of sNPF (MJ94>sNPF-Ri and sNPFc00448) yielded fat body cells with dFOXO predominantly localized in the nucleus (Fig. 4m–o, q–s). Finally, dFOXO induces expression of the translational inhibitor d4E-BP 21,22, and, consist-ent with our observations, expression of d4E-BP was elevated in animals where sNPF was inhibited (MJ94>sNPF-Ri and sNPFc00448) and reduced in animals where sNPF was overexpressed (MJ94>2XsNPF) (Fig. 4c).

Besides cell growth, Drosophila insulin-like peptides modulate aspects of metabolism and ageing. For instance, ablation of the IPCs reduces animal size, elevates the level of haemolymph carbohydrates (Fig. 1c, 5a) and extends lifespan3,4. We therefore assessed trehalose and glucose in sNPF mutant flies. As predicted, both carbohydrates were reduced upon sNPF overexpression, and both were elevated in sNPF hypomorphs (Fig. 5a). We also measured the lifespan of sNPF mutants. As expected, inhibition of sNPF by MJ94>sNPF-Ri increased median lifespan by 16–21% (Fig. 5c), whereas sNPF overexpression (MJ94>2XsNPF) did not affect lifespan in flies (Fig. 5b).

Overall, the effects on Dilp1 and Dilp2 expression in IPCs regulated by sNPF are associated with cellular, carbohydrate and lifespan responses that are predicted to be caused by changes in the actual level of available insulin peptides. We conclude that sNPF ultimately regulates insulin secretion from the IPC to affect target tissue insulin/dFOXO signalling and thus modulate growth, metabolism and lifespan (Fig. 5d).

Regulation of food consumption by neuropeptides is a critical step for interventions for managing obesity and metabolic syndromes7. Mammalian NPY is known to positively regulate appetite and has thus been thought to promote weight gain primarily by affecting food intake8,23. Here we find a novel physiological role for NPY that is conserved by sNPF of Drosophila. These neuropeptides can affect growth, metabolism and lifespan by modulating ERK-regulated transcription of insulin-like pep-tides. In Drosophila, sNPFnergic and IPC neurons are adjacent in the brain. We find, however, that pancreatic β-cells are also responsive to NPY, which is of hypothalamic origin. Although the hypothalamic neu-rosecretory cells and responding pancreatic endocrine cells are spatially distinct in mammals, recent developmental analysis suggests a paral-lel developmental pathway for hypothalamic neurosecretory cells and

the IPCs of Drosophila24, raising the possibility of a common molecular mechanism for β-cell formation. This would suggest that β-cells are not only evolutionarily tied to the hypothalamic neurosecretory cells but also that they retain their functional relationship to their hypothalamic origin by regulating insulin in response to the neuropeptide NPY.

METHODSDrosophila culture and stocks. Drosophila melanogaster were cultured and kept at 25 °C using the standard method. Wild-type Oregon-R, w–, UAS–nGFP, UAS–DsRed and UAS–reaper were obtained from the Bloomington Stock Center (Bloomington, IN), and MJ94–Gal4 was a gift from Y. D. Chung (University of Seoul, Seoul, Korea), Dilp2–Gal4 from E. Rulifson (University of California, San Francisco, CA), UAS–Dilp2 from G. H. Lee (University of Tennessee, Knoxville, TN) and UAS–rolledSEM and UAS–DMKP-3 from J. Chung (Korea Advanced Institute of Science and Technology, Daejeon, Korea). sNPFc00448 was obtained from Harvard Stock Center (Exelixis stock collection, Harvard Medical School, Boston, MA), which contains a piggyBac element in the first intron of the sNPF gene. UAS–sNPF, UAS–2XsNPF and UAS–sNPF-Ri trans-genic flies were described by Lee et al.10. UAS–sNPFR1 and UAS–sNPFR1-DN (dominant negative) transgenic flies were generated by the p-element-medi-ated germline transformation method25 with the pUAS–sNPFR1 construct containing the full length sNPFR1 cDNA or the pUAS–sNPFR1-DN construct containing the third intracellular domain (3rd ICD) of sNPFR1 cDNA in the pUAS vector. Because the 3rd ICD of G-protein-coupled receptors must bind to downstream G proteins to send a signal, overexpression of the 3rd ICD of sNPFR1 can be used as a dominant-negative mutant by masking downstream G proteins. UAS–Dilp1-Ri and UAS–Dilp2-Ri were obtained from Vienna Drosophila RNAi Center (VDRC, Vienna, Austria). Sequence information for RNAi lines is available in VDRC website (http://stockcenter.vdrc.at/). To express these UAS lines, the UAS/Gal4 system was used26.

Cell culture and the treatment of sNPF or NPY peptide. Drosophila CNS-derived neural BG2-c6 cell line, purchased from the Drosophila Genomics Resource Center (Indiana University, Bloomington, IN), were maintained at 25 °C in a 60-mm culture dish containing Schneider medium (Sigma, St Louis, MO) supplemented with 10% bovine calf serum, 50 µg ml–1 penicillin and 50 µg ml–1 streptomycin. Rat insulinoma INS-1 cells were cultured in the RPMI 1640 medium containing 2 mM l-glutamine supplemented with 10% fetal bovine serum, 5.6 mM glucose, 10 mM HEPES, 1 mM sodium pyruvate, 50 µM 2-mer-captoethanol, 100 IU ml–1 penicillin and 100 µM streptomycin in 5% CO2 at 37 °C. INS-1 cells were plated 105 cells ml–1 with the culture medium in a 12-well plate. Before peptide treatments, cells were starved for 24 h in the serum-free medium and treated with 100 nM synthetic 19-amino-acid sNPF peptide or 100 nM human NPY 1-36 peptide (Sigma, St Louis, MO). ERK-specific kinase MEK inhibitor PD98059 was purchased from Calbiochem (San Diego, CA). MEK inhibitors (20 µM) prepared in dimethylsulfoxide (DMSO) were added into the medium before treatment of sNPF or NPY peptide. The same amount of DMSO was added to the control plate.

Semi-quantitative and quantitative reverse transcription–polymerase chain reaction analysis (RT–PCR). For semi-quantitative RT–PCR analysis, adult heads or the fat body from synchronized third instar larvae (AEL 74–78 h) was dissected in PBS as described previously11. For quantitative RT–PCR analy-sis, ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster, CA) and SyberGreen PCR Core reagents (Applied Biosystems, Foster, CA) were used. mRNA levels were expressed as the relative fold change against the normalized rp49 mRNA. The comparative cycle threshold (Ct) method (User Bulletin 2, Applied Biosystems, Foster, CA) was used to analyse the data. All experiments were repeated at least three times and the data was presented as the mean and error bar (±s.e.m.). Primers used in the RT–PCR analyses were listed in Table S1.

RNA in situ hybridization in larval CNS. RNA in situ hybridization in the whole-mount third instar larval CNS with the Dig-labelled antisense RNA probe was performed as described previously27. The larval CNS was mounted in 70% glycerol. The images were acquired using the Olympus BX60 light microscope (Center Valley, PA).



l e t t e r s

Generation of sNPFR1 antiserum and immunostaining in larval CNS and fat body. sNPFR1 antiserum was generated by the immunization of rats with the synthetic peptide (ASDEDRSGGIIHN) corresponding to the amino-ter-minal extracellular domain of the sNPFR1 receptor protein. For immunos-taining, larval brain or fat bodies from third instar larvae (AEL 74–78 h) were dissected in PBS, fixed in 4% formaldehyde and blocked in 5% BSA and 10% normal goat serum. Primary antibodies were incubated overnight at 4 °C and secondary antibodies were incubated for 2 h at room temperature. The tis-sues were mounted in the DABCO solution (70% glycerol, 2.5% DABCO, Sigma, St Louis, MO) and fluorescence images were acquired by the AxioVert 200M microscope with Apotome (Carl Zeiss, Jena, Germany). sNPF (1:200), sNPFR1 (1:200), Dilp2 (1:500, a gift from E. Rulifson), dFOXO (1:500, a gift from O. Puig, University of Helsinki, Helsinki, Finland) primary antibodies, and anti-rat IgG Alexa 594, anti-rabbit IgG Alexa 488, or Alexa 594 secondary antibodies (1:200, Molecular Probes, Carlsbad, CA) were used. For visualizing the margin of fat body cells, the tissues were incubated with Phalloidin-FITC (1:1000, Sigma, St Louis, MO) for 30 min at room temperature. DAPI was used for DNA staining.

Western blot analysis. Total protein from adult heads or third instar larval fat bodies was isolated in the Drosophila homogenate buffer (10 mM HEPES, 100 mM KCl, 1 mM EDTA, 5 mM DTT, 0.1% TritonX-100, 10% glycerol and proteinase inhibitor cocktail from Sigma, St Louis, CA). Total proteins from sNPF-peptide-treated BG2-c6 or NPY-peptide-treated INS-1 cells were isolated by the PRO-PREP protein extraction buffer (iNtRON biotechnology, Seoungnam, Korea). Western blot analyses were performed as described previously10. Phospho-ERK (Sigma, St Louis, MO), Rolled, ERK1/2 (Santa Cruz, Santa Cruz, CA), phopho-Akt (Ser505, Cell signaling, Danvers, MA), Akt (Cell signaling, Danvers, MA), NPYR1, 2 (Santa Cruz, Santa Cruz, CA) and β-actin (Abcam, Cambridge, MA) primary antibodies and horseradish-peroxidase-conjugated anti-rabbit IgG (Santa Cruze, Santa Cruz, CA) and anti-mouse IgG secondary antibody (Sigma, St Louis, MO) were used.

Measurement of the size of adults, wings and fat body cells. To avoid over-crowding and food shortages, the eggs laid by five female flies for 6 h at 25 ºC were cultured. Body sizes of the 50 3–5-day-old female flies were measured from the anterior end of the head to the posterior end of the abdomen. Wing size and cell size and number in the wing were calculated as described previously3. We used the AxioVision LE Rel 4.3 software (Carl Zeiss, Jena, Germany) to measure the length of L2 vein in the wing viewed at ×100 magnification. To measure the cell size of the larval fat body, areas of single cells viewed at ×20 magnification were measured by the same AxioVision LE Rel 4.3 software. At least three experiments were performed in each assay. The data were presented as the mean and error bar (± s.e.m.). ANOVA (one-way F-test) program was used for the statistical analyses, and P<0.05 was accepted as statistically significant.

Measurement of the trehalose and glucose levels in larval haemolymph. Haemolymph from the third instar larvae (AEL 74–78h) was collected and con-centrations of trehalose and glucose were measured as described previously3. The statistical significance was tested by Microsoft Excel-based application for the student t-test statistical analysis.

Measurement of lifespan of adult flies. To measure the lifespan of sNPF mutants, newly eclosed female adults from each genotype were collected over 48 h and assigned to 1 l demography cages to a density of 100 individuals. Food vials (10% sugar and 12% yeast) were attached to each cage and changed every 2 days, at which point dead flies were removed and recorded. Three replicated cages were established for each genotype. Cages were maintained at 25 °C, 40% relative humidity with a 12 h light–dark cycle.

Note: Supplementary Information is available on the Nature Cell Biology website.

AcKnOwLedgeMenTSWe thank S.K. Ju for technical support, S.H. Hong for INS-1 cell line and E. Rulifson and O. Puig for Dilp2 and dFOXO antibodies. This work was supported by grants from Korea Research Foundation (KRF-2006-312-C00637), BioGreen 21Program of Korea Rural Development Administration (20070401034024) and KRIBB Research Initiative Program (KGM 1310811).

Published online at http://www.nature.com/naturecellbiology/ reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions

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3. Rulifson, E. J., Kim, S. K. & Nusse, R. Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science 296, 1118–1120 (2002).

4. Broughton, S.J. et al. Longer lifespan, altered metabolism, and stress resistance in Drosophila from ablation of cells making insulin-like ligands. Proc. Natl Acad. Sci. USA 102, 3105–3110 (2005).

5. Tatar, M., Bartke, A. & Antebi, A. The endocrine regulation of aging by insulin-like signals. Science 299, 1346 1351 (2003).

6. Brogiolo, W. et al. An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr. Biol. 11, 213 221 (2001).

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8. Emeson, R. B. & Morabito, M. V. Food fight: the NPY-serotonin link between aggression and feeding behavior. Sci STKE 2005, pe12 (2005).

9. Wahlestedt, C., Pich, E.M., Koob, G.F., Yee, F. & Heilig, M. Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science 259, 528–531 (1993).

10. Lee, K. S., You, K. H., Choo, J. K., Han, Y. M. & Yu, K. Drosophila short neuropeptide F regulates food intake and body size. J. Biol. Chem. 279, 50781–50789 (2004).

11. Feng, G. et al. Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster. Eur. J. Neurosci. 18, 227–238 (2003).

12. Chung, Y. D., Zhu, J., Han, Y. & Kernan, M. J. nompA encodes a PNS-specific, ZP domain protein required to connect mechanosensory dendrites to sensory structures. Neuron 29, 415–428 (2001).

13. Joiner, M. A. & Griffith, L. C. Mapping of the anatomical circuit of CaM kinase-depend-ent courtship conditioning in Drosophila. Learn. Mem. 6, 177–192 (1999).

14. Colombani, J. et al. A nutrient sensor mechanism controls Drosophila growth. Cell 114, 739–749 (2003).

15. Hwangbo, D. S., Gershman, B., Tu, M. P., Palmer, M. & Tatar, M. Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body. Nature 429, 562–566 (2004).

16. Wang, M. C., Bohmann, D. & Jasper, H. JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling. Cell 121, 115–125 (2005).

17. Cho, Y. R. & Kim, C. W. Neuropeptide Y promotes beta-cell replication via extracellular signal-regulated kinase activation. Biochem. Biophys. Res. Commun. 314, 773–780 (2004).

18. Kim, M. et al. MKP-3 has essential roles as a negative regulator of the Ras/mitogen-activated protein kinase pathway during Drosophila development. Mol. Cell. Biol. 24, 573–583 (2004).

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s u p p l e m e n ta ry i n f o r m at i o n

www.nature.com/naturecellbiology 1

Figure S1 sNPF mutant wings and their cell sizes and numbers. (a) Wing size was larger in MJ94>2XsNPF and smaller in sNPFc00448 than the wild-type control. The scale bar is 100 mm. (b, c) The number and size of cells were increased in MJ94>2XsNPF wing and decreased in sNPFc00448 wing

compared to the wild-type control. The ANOVA test was used to compare the genotypes: (b) WT and MJ94>2XsNPF, P = 0.005; WT and sNPFc00448, P = 0.003, (c) WT and MJ94>2XsNPF, P = 0.0001; WT and sNPFc00448, P = 3.8x10-5. The error bars represent S.E.M. and n = 20 in all genotypes.

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Figure S2 Expression levels of sNPF, Dilp3, and Dilp5 in sNPF mutants. (a) Expression level of sNPF was 3.5 times in MJ94>2XsNPF, half in MJ94>sNPF-Ri, and less than half in sNPFc00448 compared to the wild-type control. (b) Expression level of

Dilp3 was around half in MJ94>sNPF-Ri and sNPFc00448 compared to the wild-type control. (c) Expression level of Dilp5 was not affected by sNPF mutants. The error bars represent S.E.M. from three independent experiments.

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Figure S3 sNPFR1 localization in the third instar larval brain. (a) sNPFR1 antibody stained in the plasma membrane of IPC (green, arrows). (b) Dilp2>DsRed marked IPC (red, arrows). (c) sNPFR1 antibody staining in the Dilp2>DsRed larval brain showed the localization of sNPFR1 in the

plasma membrane of IPC (yellow, arrows). (d) In the third instar larval CNS, sNPFR antibody stained insulin producing cells (A), neurons in the brain hemisphere (B), sub-esophagus ganglion (C), ventral abdominal neurons (D), and descending axons in the ventral ganglion (E). Scale bars are 100 µm.

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Figure S4 Antibody staining against Dilp2 (red) and phospho-activated ERK (dp-ERK, green) in the adult brain and their intensity profiles. (a) In the control adult brain, Dilp2 antibody stained IPC and showed as the peaks in the intensity profile while dp-ERK was a basal level. (b) In the adult brain of

sNPFR1 over-expression, dp-ERK was detected in IPC (yellow, arrows) and the intensity of dp-ERK was higher than that of the control. The peaks of dp-ERK also matched with those of Dilp2. (c) In the adult brain of sNPFR1 inhibition, dp-ERK was a basal level in IPC. The scale bar is 100 µm.

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Figure S5 sNPFRl and NPYR receptors were endogenously expressed in Drosophila BG2-c6 and rat INS-1 cells, respectively. (a) sNPFR1 mRNA was expressed in the sNPF peptide treated and non-treated Drosophila BG2-c6 cells. (b) sNPFR1 receptors were concentrated on the cell

surface of BG2-c6 cells (green). DAPI stained DNA in nuclei (blue). ( c) NPYR1 and 2 receptor mRNA and proteins were expressed in the NPY peptide treated and non-treated rat insulinoma INS-1 cells. The scale bar is 20 µm.

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Supplementary Table 1 PCR Primers used in this study.

PCR primer product size sequences

sNPF 188 bp forward : CCCGAAAACTTTTAGACTCAreverse : TTTTCAAACATTTCCATCGT

sNPFR1 476 bp forward : CGAACAACAACATCATCAACreverse : ACTCGTACTCGTCTCCAGAA

Dilp1 160 bp forward : GGGGCAGGATACTCTTTTAGreverse : TCGGTAGACAGTAGATGGCT

Dilp2 183 bp forward : GTATGGTGTGCGAGGAGTATreverse : TGAGTACACCCCCAAGATAG

Dilp3 216 bp forward : AAGCTCTGTGTGTATGGCTTreverse : AGCACAATATCTCAGCACCT

Dilp5 211 bp forward : AGTTCTCCTGTTCCTGATCCreverse : CAGTGAGTTCATGTGGTGAG

d4EBP 457 bp forward : GATCACCAGGAAGGTTGTCreverse : GGTCAATATGACCGAGAGAA

rp49 122 bp forward : AGGGTATCGACAACAGAGTGreverse : CACCAGGAACTTCTTGAATC

NPY R1 606 bp forward : TACTGTCATTTGGGTACTGGCGGTreverse : TTGGACACGTCCGTATGCATGGTA

NPY R2 459 bp forward : AGGTGCAGAGGCAGATGAGAATCAreverse : TCCAGGTGGTAGACAATGCAACGA

GAPDH 139 bp forward : GTATTGGGCGCCTGGTCACCreverse : CGCTCCTGGAAGATGGTGATGG

Insulin1 209 bp forward : TGTCAAACAGCACCTTTGTGGTCCreverse : ACTGATCCACAATGCCACGCTTCT

Insulin2 301 bp forward : GCCCTGTGGATCCGCTTCCTreverse : AGAGAGCAGATGCTGGTGCAG

Table S1


npfr1 nat cell biol

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