Systemic corazonin signalling modulates stress responses and metabolism in Drosophila

Abstract

Stress triggers cellular and systemic reactions in organisms to restore homeostasis. For instance, metabolic stress, experienced during starvation, elicits a hormonal response that reallocates resources to enable food search and readjustment of physiology. Mammalian gonadotropin-releasing hormone (GnRH) and its insect orthologue, adipokinetic hormone (AKH), are known for their roles in modulating stress-related behaviour. Here we show that corazonin (Crz), a peptide homologous to AKH/GnRH, also alters stress physiology in Drosophila The Crz receptor (CrzR) is expressed in salivary glands and adipocytes of the liver-like fat body, and CrzR knockdown targeted simultaneously to both these tissues increases the fly's resistance to starvation, desiccation and oxidative stress, reduces feeding, alters expression of transcripts of Drosophila insulin-like peptides (DILPs), and affects gene expression in the fat body. Furthermore, in starved flies, CrzR-knockdown increases circulating and stored carbohydrates. Thus, our findings indicate that elevated systemic Crz signalling during stress coordinates increased food intake and diminished energy stores to regain metabolic homeostasis. Our study suggests that an ancient stress-peptide in Urbilateria evolved to give rise to present-day GnRH, AKH and Crz signalling systems.

Keywords: corazonin receptor; fat body; insulin-like peptides; neuropeptide; stress signalling.

Figure 1.

The corazonin receptor (CrzR) is expressed in adipocytes of D. melanogaster fat body and its knockdown leads to a compensatory increase of corazonin peptide (Crz) and transcript (crz) expression. (a) The CrzR is expressed in the adult fly fat body (CrzR>GFP), mainly in the abdomen. (b,c) Two different Gal4 drivers (ppl-Gal4 and to-Gal4) display GPF expression in adult flies. These were used for targeting UAS-CrzR-RNAi in subsequent experiments. In electronic supplementary material, figure S1a–d, we show details of GFP expression, as well as GFP expression in other tissues. (d) Expression of CrzR in salivary gland revealed by CrzR-Gal4>UAS-GFP). (e) CrzR-RNAi efficiency was tested by qPCR in ppl>CrzR-RNAi flies (ppl>CrzR-Ri). The efficiency for the CrzR-RNAi driven by an actin-Gal4 is shown in electronic supplementary material, figure S1e. (f) Corazonin-immunolabelling is found in a set of seven dorsolateral peptidergic neurons (DLPs) in each brain hemisphere (shown in w¹¹¹⁸ fly). (g–i) Knockdown of CrzR in fat body/salivary glands by ppl-Gal4 induces an increase of CRZ expression in DLPs. (j,k) Using ppl-Gal4 and to-Gal4 to drive CrzR-RNAi results in increased crz transcript levels, especially after 36 h starvation. Data in graphs are presented as means ± s.e.m., n = 3–4 independent replicates with 8–12 flies in each replicate. (e) Kruskal–Wallis's test followed by pairwise comparisons using Wilcoxon's rank sum test with ^#p < 0.05, ^##p < 0.01, (i) Student's t-test with *p < 0.05, **p < 0.01 and (j,k) ANOVA followed with Tukey's test p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Knockdown of CrzR in the fat body increases stress resistance in flies. We used 3-day-old male flies for all experiments. All results were analysed by log-rank test and data for flies with CrzR-knockdown (CrzR-Ri) were compared with their respective controls. Data are presented as means ± s.e.m. (n = 80–100 flies for each genotype, run in four replicates). (a,b) Flies with CrzR knockdown targeted to adipocytes displayed increased survival at starvation (water, but no food; χ² = 35.5, p < 0.0001 and χ² = 22.3, p < 0.0001). (c,d) Knockdown of CrzR with to-Gal4 results in enhanced survival under desiccation (no food, no water; χ² = 48.6, p < 0.0001), whereas the same manipulation using ppl-Gal4 does not affect survival (χ² = 3.8, p = 0.0519). (e,f) to-Gal4-driven CrzR-RNAi drastically increased oxidative stress resistance (food supplemented with 10 mM paraquat; χ² = 71, p < 0.0001) and a similar phenotype was observed with ppl-Gal4-driven CrzR-RNAi (χ² = 11.6, p < 0.001).

Figure 3.

CrzR-knockdown targeted to the periphery increases stored carbohydrates in flies exposed to 36 h of starvation. (a,b) In flies with CrzR-RNAi targeted to fat body (ppl>CrzR-Ri and to>CrzR-Ri) concentrations of body glucose are about twofold higher than in controls after 36 h starvation, but CrzR-knockdown has no effect in fed flies. (c,d) CrzR-knockdown in fat body results in higher levels of whole body trehalose after starvation than in control flies, but in fed flies no significant difference is seen. (e,f) Glycogen stores are depleted by starvation, but flies with CrzR-Ri targeted to adipocytes contain more glycogen in both experimental conditions used. Data are presented as means ± s.e.m., n = 4 replicates with 10–15 flies in every replicate (*p < 0.05, **p < 0.01 and ***p < 0.001; ANOVA followed by Tukey's test, or ^#p < 0.05 (Kruskal–Wallis's test followed by pairwise comparisons using Wilcoxon's rank sum test). See also electronic supplementary material, figure S3 for graphs with circulating carbohydrates and whole body triacylglycerides (TAG).

Figure 4.

Flies with CrzR-knockdown targeted to adipocytes/salivary glands feed less. Accumulated food intake over 4 days of 3-day-old male flies (µl 96 h per fly) was estimated by CAFE assay. Data are presented as means ± s.e.m., n = 3 replicates with 10 flies in each replicate (*p < 0.05, **p < 0.01 from the indicated group as assessed by ANOVA, followed by Tukey's test).

Figure 5.

CrzR-knockdown targeted to fat body/salivary gland differentially affects levels of dilp transcripts and AKH peptide in fed and starved flies. (a) Starvation leads to a strong decrease of dilp2 mRNA for all genotypes, but is not affected by CrzR-RNAi. (b) dilp3 expression is lower in both ppl>CrzR-Ri and to>CrzR-Ri flies (compared with controls) in both normal and starvation conditions. (c) dilp5 mRNA expression is very low after starvation, but a higher dilp5 level in CrzR-Ri compared with controls was seen only under normal feeding conditions. (d) The DILP2 immunolabelling intensity in IPCs was not affected by CrzR-knockdown in fat body. (e) The DILP5 immunolabelling intensity in IPCs was also not affected by CrzR-RNAi. (f) The Akh transcript level was not affected by CrzR-RNAi in fat body with either driver. (g,h) The AKH peptide level as measured by immunolabelling in corpora cardiaca was drastically reduced in flies with CrzR-knockdown in fat body. In these graphs, data are presented as means ± s.e.m., n = 3–4 independent replicates with 10–15 flies in each replicate (p < 0.05, **p < 0.01, ***p < 0.001; ANOVA followed with Tukey's test).

Figure 6.

Changes in expression of gene transcript in dissected fat body determined by qPCR in ppl>CrzR-RNAi flies. (a) The dilp6 transcript is not affected in ppl>CrzR-Ri flies. (b) The cytokine unpaired-2 (Upd2) mRNA is reduced in the fat body. (c) The brummer TAG lipase (encoded by bmm) transcript is decreased. (d) Expression of the transcript of phosphoenolpyruvate carboxykinase (encoded by pepck) is not affected. (e) No change was detected in mRNA of Superoxide dismutase 2 (Sod2). (f) The stress responsive gene Neural Lazarillo (Nlaz) increased after CrzR-RNAi. (g) Another stress inducible gene, Turandot A (TotA), was strongly activated in fat body of flies with CrzR-RNAi. The expression levels in dissected abdominal fat body were calculated with the 2^−ΔΔCt method relative to the control ppl>w¹¹¹⁸, which is set at one. Data are presented as means ± s.e.m., n = 3 independent replicates with 20–30 fat body samples in each replicate Kruskal–Wallis's test followed by pairwise comparisons using Wilcoxon's rank sum test with (^#p < 0.05, ^##p < 0.01).

Figure 7.

Summary of changes in gene transcripts measured from whole flies after CrzR-RNAi with two different fat body Gal4 drivers. Knockdown of the CrzR in fat body does not induce a strong response in gene transcription at the level of whole flies during normal (fed) conditions. Data from qPCR are shown as average fold-changes of expression compared with level in the respective controls during normal conditions (see key for colour coding). Both experimental fly crosses, ppl>CrzR-Ri and to>CrzR-Ri, display similar patterns of gene transcript changes. Thus, TotA was upregulated during normal conditions in flies with CrzR-knockdown, but starvation results in reduced TotA. dilp6 mRNA is not affected in whole flies by any condition. Starvation leads to downregulation of Upd2, Sod2 and Nlaz, whereas bmm and pepck expression increases. In electronic supplementary material, figure S6, these experiments are shown in regular graphs.

Figure 8.

CrzR-knockdown with a CrzR-Gal4 driver generates phenotypes similar to peripheral receptor knockdown. (a) Knockdown efficiency of CrzR-RNAi with the CrzR-Gal4. Data are presented as means ± s.e.m., n = 4 independent replicates with 10–15 flies in each replicate (***p < 0.001, ANOVA followed with Tukey's test). (b) Resistance to oxidative stress induced by paraquat feeding increases after CrzR-RNAi (χ² = 137 and 375, p < 0.0001 comparing with the Gal4 and UAS control, respectively). (c) Food intake is reduced after CrzR-RNAi. Accumulated food intake over 4 days of 3-day-old male flies (µl 96 h per fly) was estimated by CAFE assay. Data are presented as means ± s.e.m., n = 4 independent replicates with eight flies in each replicate (p < 0.05, **p < 0.01, ANOVA followed with Tukey's test).

Figure 9.

Crz knockdown with a Crz-Gal4 driver generates phenotypes similar to peripheral receptor knockdown. (a) Knockdown efficiency of Crz-RNAi with the Crz-Gal4. Data are presented as means ± s.e.m., n = 4 independent replicates with 10 flies in each replicate (***p < 0.001, ANOVA followed with Tukey's test). (b) Resistance to desiccation stress is increased after Crz-RNAi. (c) Resistance to oxidative stress induced by paraquat feeding increases after Crz-RNAi (χ² = 54 and 137, p < 0.0001 comparing with the Gal4 and UAS control, respectively). (d) Food intake is reduced after Crz-RNAi. Accumulated food intake over 4 days of 3-day-old male flies (µl 96 h per fly) was estimated by CAFE assay (n = 4 independent replicates with eight flies in each replicate; ***p < 0.001, ANOVA followed with Tukey's test).