Reduction of DILP2 in Drosophila triages a metabolic phenotype from lifespan revealing redundancy and compensation among DILPs

Abstract

The insulin/IGF-like signalling (IIS) pathway has diverse functions in all multicellular organisms, including determination of lifespan. The seven insulin-like peptides (DILPs) in Drosophila are expressed in a stage- and tissue-specific manner. Partial ablation of the median neurosecretory cells (mNSCs) in the brain, which produce three DILPs, extends lifespan, reduces fecundity, alters lipid and carbohydrate metabolism and increases oxidative stress resistance. To determine if reduced expression of DILPs is causal in these effects, and to investigate possible functional diversification and redundancy between DILPs, we used RNA interference to lower specifically the transcript and protein levels of dilp2, the most highly expressed of the mNSC-derived DILPs. We found that DILP2 was limiting only for the increased whole-body trehalose content associated with mNSC-ablation. We observed a compensatory increase in dilp3 and 5 mRNA upon dilp2 knock down. By manipulation of dfoxo and dInR, we showed that the increase in dilp3 is regulated via autocrine insulin signaling in the mNSCs. Our study demonstrates that, despite the correlation between reduced dilp2 mRNA levels and lifespan-extension often observed, DILP2 reduction is not sufficient to extend lifespan. Nor is the increased trehalose storage associated with reduced IIS sufficient to extend lifespan. To understand the normal regulation of expression of the dilps and any functional diversification between them will require independent control of the expression of different dilps.

Figure 1. Characterization of DILP2 knock-down in UAS-dilp2RNAi/d2GAL flies.

The effect of UAS-dilp2RNAi driven by d2GAL in the mNSCs on dilp expression in 7 day old adult female heads was measured by quantitative RT-PCR and Western blot analysis. (A) dilp 2, 3 and 5 relative transcript levels in dilp2RNAi/d2GAL and control female heads. (B) Western blot analysis of DILP2 (top panel) or the tubulin loading control (bottom panel) in total head protein extracted from females of the indicated genotype. (C) dilp 2, 3 and 5 relative transcript levels in d2GAL/UAS-InR^DN and control female heads. (D) dilp 2, 3 and 5 relative transcript levels in a FOXO null mutant (FOXO^21/25) and control female heads. (A, C and D): data are shown as mean relative expression±SEM (N = 5), * denotes significant difference to controls (P<0.05).

Figure 2. dilp2RNAi/d2GAL mated females are not long-lived or less fecund than controls.

Survival curves, median lifespans, percentage increase compared to control, and sample sizes are as follows. (A): Experiment 1. Median lifespan of: UAS-rpr/d2GAL = 75 days (13.6% increase over UAS-rpr/+ & d2GAL/+ controls, P<0.0001), N = 69; UAS-dilp2RNAiA/d2GAL = 67 days, N = 71; UAS-dilp2RNAiA/+ = 66 days, N = 36; UAS-dilp2RNAiB/d2GAL = 66 days, N = 130; UAS-dilp2RNAiB/+ = 60 days, N = 92; UAS-rpr/+ = 66 days, N = 110; and d2GAL/+ = 66 days, N = 61. (B) Experiment 2. Median lifespan of: UAS-rpr/d2GAL = 81 days (19% increase over UAS-rpr/+ control, P<0.0001), N = 155; UAS-dilp2RNAiA/d2GAL = 76 days, N = 157; UAS-dilp2RNAiA/+ = 73 days, N = 137; UAS-dilp2RNAiB/d2GAL = 71 days, N = 145; UAS-dilp2RNAiB/+ = 69 days, N = 129; UAS-rpr/+ = 68 days, N = 139; and d2GAL/+ = 64 days, N = 153. (C) Experiment 3. Median lifespan of: UAS-rpr/d2GAL = 73 days (12% increase over d2GAL/+ control, P<0.0001), N = 153; UAS-dilp2RNAiA/d2GAL = 68 days, N = 180; UAS-dilp2RNAiA/+ = 67 days, N = 166; UAS-dilp2RNAiB/d2GAL = 65 days, N = 179; UAS-dilp2RNAiB/+ = 67 days, N = 175; UAS-rpr/+ = 59 days, N = 149; and d2GAL/+ = 65 days, N = 169. (D) Fecundity of females from experiment 1 shown in (A). (E) Fecundity of females from experiment 2 shown in (B). Data are shown as mean number of eggs laid per female per day±SEM.

Figure 3. Survival of dilp2RNAi/d2GAL female flies under oxidative stress and starvation.

Survival curves, median lifespans, percentage increase compared to control, and sample sizes of 7 day old mated females are as follows: (A) Experiment 1 on 5% H₂O₂. Median lifespans (in days) of: UAS-rpr/d2GAL = 4.04, N = 104, (34% increase over d2GAL/+ control, P<0.0001); UAS-dilp2RNAiA/d2GAL = 3.25, N = 102; UAS-dilp2RNAiA/+ = 3.25, N = 100; UAS-dilp2RNAiB/d2GAL = 3.25, N = 77; UAS-dilp2RNAiB/+ = 2.9, N = 79; UAS-rpr/+ = 3.01, N = 82; and d2GAL/+ = 3.01, N = 98. (B) Experiment 2 on 5% H₂O₂. Median lifespans (in days) of: UAS-rpr/d2GAL = 3.85, N = 77, (15.6% increase over d2GAL/+ control, P<0.0001); UAS-dilp2RNAiA/d2GAL = 3.21, N = 83; UAS-dilp2RNAiA/+ = 3, N = 96; UAS-dilp2RNAiB/d2GAL = 3.33, N = 58; UAS-dilp2RNAiB/+ = 3.21, N = 84; UAS-rpr/+ = 3.21, N = 91; and d2GAL/+ =  3.33, N = 87. (C–E) Experiment 1 on 1% agar. Median lifespans (in days) of: UAS-rpr/d2GAL (mNSC-ablated) = 5.9, N = 120, (47.5% increase over UAS-rpr/+ & d2GAL/+ controls, P<0.0001); UAS-dilp2RNAiA/d2GAL = 4.17, N = 112, (4.25% increase over UAS-Dilp2RNAiA/+ control, P<0.0001); UAS-dilp2RNAiA/+ = 4, N = 115; UAS-dilp2RNAiB/d2GAL = 4.17, N = 98, (4.25% increase over d2GAL/+ control, P = 0.0004); UAS-dilp2RNAiB/+ = 3.86, N = 117; UAS-rpr/+ = 4, N = 116; and d2GAL/+ = 4, N = 117. (F–H) Experiment 2 on 1% agar, Median lifespans (in days) of: UAS-rpr/d2GAL = 6.2, N = 91, (25% increase over UAS-rpr/+ control, P<0.0001); UAS-Dilp2RNAiA/d2GAL = 4.96, N = 94, (19.5% increase over UAS-dilp2RNAiA/+ control, P = 0.0041); UAS-dilp2RNAiA/+ = 4.15, N = 91; UAS-dilp2RNAiB/d2GAL = 4.15, N = 88; UAS-dilp2RNAiB/+ = 3.96, N = 91; UAS-rpr/+ = 4.96, N = 98; and d2GAL/+ = 3.96, N = 98. (I–K) Experiment 3 on 1% agar, Median lifespans (in days) of: UAS-rpr/d2GAL = 4.85, N = 89, (24% increase over UAS-rpr/+ control, P<0.0001); UAS-dilp2RNAiA/d2GAL = 3.25, N = 85; UAS-dilp2RNAiA/+ = 3.17, N = 94; UAS-dilp2RNAiB/d2GAL = 3.9, N = 76, (23% increase over d2GAL/+ control, P<0.0001); UAS-dilp2RNAiB/+ = 3.06, N = 76; UAS-rpr/+ = 3.9, N = 85; and d2GAL/+ = 3.17, N = 82.

Figure 4. The effect of dilp2RNAi expression in mNSCs on hemolymph glucose and trehalose levels, and whole-body trehalose, glycogen and lipid content.

(A) Hemolymph glucose and trehalose concentrations in 7 day old mated females, maintained on standard food with 100 g/l of sugar and fasted on 1% agar for 5 hours prior to testing. N = 10 for each genotype. (B) Hemolymph glucose and trehalose concentrations in third instar wandering larvae that developed on standard food with 100 g/l of sugar: UAS-rpr/d2GAL (N = 7), UAS-dilp2RNAiA/d2GAL (N = 7), UAS-dilp2RNAiA/+ (N = 5), UAS-dilp2RNAiB/d2GAL (N = 6), UAS-dilp2RNAiB/+ (N = 6), UAS-rpr/+ (N = 5) and d2GAL/+ (N = 6). (C) Whole-fly trehalose content per mg of fly (fresh weight). UAS-rpr/d2GAL (N = 20), UAS-dilp2RNAiA/d2GAL (N = 20), UAS-dilp2RNAiA/+ (N = 10), UAS-dilp2RNAiB/d2GAL (N = 20), UAS-dilp2RNAiB/+ (N = 10), UAS-rpr/+ (N = 10) and d2GAL/+ (N = 20). (D) Glycogen content per mg of fly (fresh weight), N = 21. (E) Lipid content per mg of fly (fresh weight), N = 17. In all panels, data are shown as mean±SEM, * indicates significant difference to appropriate controls (P<0.05).