Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor

Abstract

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.

Fig. 1

Over-expression of Imp-L2 with heatshockGAL4 driver. The flies were reared at 25°C and harvested on day 7. (A) The transcript levels of Imp-L2 were determined by qPCR, normalised to Act mRNA and the ratio in hsGAL4 set to one. Means and standard errors are shown with n = 6 for hsGAL4 > UAS-Imp-L2 and n = 7 for the two controls. The measurements for hsGAL4 > UAS-Imp-L2 were compared to those for hsGAL4 by t-test: P = 0.02, to UAS-Imp-L2: P = 0.05. (B) The levels of IMP-L2 protein were determined in whole flies by western blot, with actin as the loading control. The averages and standard errors of three independent measurements of IMP-L2 protein normalised to actin are given below the images, with the average of pooled controls set to one. The levels in hsGAL4 > UAS-Imp-L2 were significantly different from the pooled controls (P = 0.04) or UAS-Imp-L2 (P = 0.01) but not from hsGAL4, by t-test. (C) Levels of IMP-L2 protein were determined in haemolymph by western blot. All flies carried the hsGAL4 driver with or without UAS-Imp-L2 and were either kept at 25°C or placed at 37°C for 2 h prior to collection of haemolymph. Full genotypes of the flies tested were: w⁻/w⁻; hsGAL4/+; +/+, w⁻/w⁻; +/+; UAS-Imp-L2/+, w⁻/w⁻; hsGAL4/+; UAS-Imp-L2/+. Note that in A and B, the differences between the two controls were not statistically significant.

Fig. 2

Increase in Imp-L2 induces 4E-BP transcript. (A) The phosphorylation of dFOXO in flies over-expressing Imp-L2. The phosphorylation was monitored as retardation on SDS-PAGE and dFOXO bands revealed by western blotting with an anti-dFOXO antibody. In separate wild-type extracts, the slower migrating band (dFOXOppp) was shown to disappear on treatment with calf intestinal phosphatase (data not shown). (B) The transcript levels of 4E-BP were determined by qPCR, normalised to Act mRNA and the ratio in hsGAL4 set to one. Means and standard errors are shown with n = 6 for hsGAL4 > UAS-Imp-L2 and n = 7 for the two controls, P < 10⁻³ to either control by t-test, while the differences between the two controls were not significant. Full genotypes of the flies tested were: w⁻/w⁻; hsGAL4/+; +/+, w⁻/w⁻; +/+; UAS-Imp-L2/+, w⁻/w⁻; hsGAL4/+; UAS-Imp-L2/+.

Fig. 3

IMP-L2 binds DILP2 and DILP5. (A) Far-western blot was performed on head proteins from flies of the indicated genotypes using S2-cell conditioned media containing either IMP-L2-myc₆ (right-hand panel) or not (mock; left-hand panel) and the binding of IMP-L2-myc₆ visualised by an anti-myc antibody. Bands corresponding to DILP2 and DILP5 are indicated. (B) Higher exposure of the far-western shown above in A. (C) Tubulin used as loading control. Note that in all the panels the lanes shown align with the genotypes indicated in A. Full genotypes of the flies tested were: w⁻/w⁻; +/+; +/+, w⁻/w⁻; +/+; dilp2Δ/dilp2Δ, w⁻/w⁻; +/+; dilp3Δ/dilp3Δ, w⁻/w⁻; +/+; dilp5Δ/dilp5Δ.

Fig. 4

Increase in Imp-L2 results in increased dilp2, dilp3 and dilp5 transcription. The levels of dilp2, dilp3 and dilp5 transcripts were determined by qPCR in whole fly RNA, normalised to Act mRNA and the average ratio of the two control genotypes set to one for each transcript. Means and standard errors are shown with n = 4 for all measurements except for dilp5 in UAS-Imp-L2 where n = 3. Two-way anova showed that the effect of genotype was significant: P < 10⁻⁴, where hsGAL4 > UAS-Imp-L2 was different to both controls by t-test while the differences between the two controls were not significant, and there was no significant interaction between genotype and transcript. Full genotypes of the flies tested were: w⁻/w⁻; hsGAL4/+; +/+, w⁻/w⁻; +/+; UAS-Imp-L2/+, w⁻/w⁻; hsGAL4/+; UAS-Imp-L2/+.

Fig. 5

Over-expression of Imp-L2 increases lifespan, oxidative stress resistance and whole-fly lipid content, while reducing fecundity. (A) Means and standard errors of the measurements of whole-fly triacylglycerol (TAG) per protein are shown, with n = 8 for each genotype, where hsGAL4 > UAS-Imp-L2 is different to both controls by t-test (P = 0.01 to hsGAL4, P = 0.003 to UAS-Imp-L2). (B) The average number of eggs laid per female over 24 h was measured in ten separate vials per genotype at the times indicated. Means and standard errors are shown. Two Way anova showed that the effect of genotype was significant: P = 0.008, where hsGAL4 > UAS-Imp-L2 is different to both controls by t-test; effect of time: P < 10⁻⁴; no interaction of the two main effects. Estimate of the cumulative eggs laid per female: 72 for hsGAL4, 72 for UAS-Imp-L2 and 60 for hsGAL4 > UAS-Imp-L2. (C) Five-day old female flies (hsGAL4 > UAS-Imp-L2 n = 59, hsGAL4 n = 99, UAS-Imp-L2 n = 99) were placed on food containing 5% H₂O₂/suchrose and their survival determined over time. The survival of hsGAL4 > UAS-Imp-L2 was significantly different from either control by Log-rank test (P < 10⁻⁴). (D) Lifespans of hsGAL4 > UAS-Imp-L2 (n = 137, med = 71 days, max = 74 days) and the two genetic controls hsGAL4 and UAS-Imp-L2 (n = 142, med = 61 days, max = 74 days and n = 139, med = 61 days, max = 74 days). The survival of hsGAL4 > UAS-Imp-L2 was significantly different from either control by Log-rank test, P < 10⁻⁴. Note in B, C and D the same symbols are used for the genotypes and are indicated in B. Full genotypes of the flies tested were: w⁻/w⁻; hsGAL4/+; +/+, w⁻/w⁻; +/+; UAS-Imp-L2/+, w⁻/w⁻; hsGAL4/+; UAS-Imp-L2/+. Note that in all panels the differences between the two controls were not statistically significant.

Fig. 6

Adult-onset induction of Imp-L2 increases lifespan. (A) Lifespans of female ActGS > UAS-Imp-L2 flies induced to ubiquitously over-express Imp-L2 by feeding RU486-containing food from day 3 of adulthood (+RU486, n = 143, med = 75 days, max = 84) or the uninduced controls (−RU486, n = 143, med = 63 days, max = 79 days). The survival of ActGS > UAS-Imp-L2+ RU486 was significantly different from the −RU486 control by Log-rank test (P < 10⁻⁴). Maximal lifespan was also extended upon Imp-L2 over-expression (P < 0.05 by Log-rank test on the final 10% survivors). (B) Levels of Imp-L2, 4E-BP and dilp2 mRNA relative to Act mRNA were determined by qPCR in ActGS > UAS-Imp-L2 female flies after 4 days of induction or in the uninduced control. Means and standard errors are shown, with values for the uninduced control set to 1 and with n = 7 for all measurements except for Imp-L2 mRNA in +RU486 where n = 6. In each case −RU486 was significantly different to +RU486 by t-test (Imp-L2: P < 10⁻⁴, dilp2: P = 5 × 10⁻⁴, 4E-BP: P = 0.04). (C) The levels of IMP-L2 protein were determined in whole flies (top) or in haemolymph (bottom) by western blot. For whole-fly extracts, actin was used as the loading control, and the averages and standard errors of three independent measurements of IMP-L2 protein normalised to actin are given below the images, with the levels in the uninduced control set to one; these were different by t-test (P = 0.04). Full genotype of ActGS > UAS-Imp-L2 flies: w⁻/w⁻; ActGS/+; UAS-Imp-L2/+.

Fig. 7

Tissue-restricted over-expression of Imp-L2 increases lifespan. (A) Lifespans of female flies over-expressing Imp-L2 in the mNSC (dilp2GAL4 > UAS-Imp-L2, W^dahn = 142, med = 71 days, max = 77 days; w¹¹¹⁸n = 141, med = 65 days, max = 72 days) and the two genetic controls (dilp2GAL4, W^dahn = 149, med = 66 days, max = 74 days; w¹¹¹⁸n = 140, med = 59 days, max = 67 days; UAS-Imp-L2, W^dahn = 142, med = 66 days, max = 74 days; w¹¹¹⁸n = 142, med = 59 days, max = 67 days; top two panels) or female flies in which over-expression of Imp-L2 in the gut and fat body by the S₁106 driver was induced at day 3 of adulthood by RU486 (S₁106 > UAS-Imp-L2+ RU486, W^dahn = 143. med = 69 days, max = 77 days; w¹¹¹⁸n = 144, med = 65 days, max = 74 days) or the uninduced controls (S₁106 > UAS-Imp-L2−RU486, W^dahn = 143, med = 62, max = 74; w¹¹¹⁸n = 150, med = 63 days, max = 70 days; lower two panels), in outbred W^dah (two panels on the left) and the inbred w¹¹¹⁸ (two panels on the right). The survival of dilp2GAL4 > UAS-Imp-L2 was significantly different from the two controls in both backgrounds by Log-rank test (P < 10⁻⁴). Log-rank test also showed the survival of S₁106 > UAS-Imp-L2 significantly altered by addition of RU486, with P < 10⁻⁶ for W^dah, P = 0.003 for w¹¹¹⁸. In all cases, the maximal lifespan was extended upon Imp-L2 over-expression (P < 0.05 by Log-rank test on the final 10% survivors). (B) IMP-L2 was measured in the haemolymph of dilp2GAL4 > UAS-Imp-L2 female flies and the two genetic controls, or in S₁106 > UAS-Imp-L2 female flies fed or not with RU486, by western blotting. (C) IMP-L2 was visualised with immunofluorescence in the guts of S₁106 > UAS-Imp-L2 female flies fed or not with RU486. IMP-L2 is indicated in red, DAPI-stained nuclei in blue. Note the red fluorescence of the gut contents. The numbers next to the images give the mean and standard error of relative fluorescence intensity per unit area of gut as quantified, after background subtraction, from at least three animals (P = 0.01 by t-test). (D) IMP-L2 was visualised with immunofluorescence in the mNSC of wandering third-instar dilp2GAL4 > UAS-Imp-L2 larvae or the two genetic controls. mNSC were identified with an anti-DILP5 antibody (green), IMP-L2 is indicated in red, DAPI in blue. The numbers next to the images give the mean and standard error of relative fluorescence intensity per mNSC quantified, after background subtraction, and averaged over at least three cells from four animals (n = 4, t-test dilp2GAL4 > UAS-Imp-L2 to dilp2GAL4 P = 0.04, to UAS-Imp-L2 P = 0.001). (E) Levels of dilp2 mRNA relative to Act mRNA were determined in the flies of the indicated genotypes/treatments, with n = 6 and the levels set to 1 in relevant controls. The levels in S₁106 > UAS-Imp-L2 were significantly altered by addition of RU486 (t-test: P = 0.05). Full genotypes of the flies used: w⁻/w⁻; +/+; dilp2GAL4/+, w⁻/w⁻; +/+; UAS-Imp-L2/+, w⁻/w⁻; +/+; dilp2GAL4/UAS-Imp-L2, w⁻/w⁻; S₁106/+; UAS-Imp-L2/k. Note that in all cases the differences between dilp2GAL4 and UAS-Imp-L2 controls were not statistically significant.