Drosophila insulin-like peptide-6 (dilp6) expression from fat body extends lifespan and represses secretion of Drosophila insulin-like peptide-2 from the brain

Abstract

Reduced insulin/IGF signaling extends lifespan in diverse species, including Drosophila melanogaster where the genome encodes seven insulin-like peptides (dilp1-7). Of these, reduced dilp2 expressed in the brain has been associated with longevity assurance when over-expression of dfoxo in fat bodies extends lifespan. Here, we show that the insulin-regulated transcription factor dFOXO positively modulates dilp6 mRNA in adult fat body. Over-expression of dilp6 in adult fat body extends lifespan and increases longevity-associated metabolic phenotypes. Adult fat body dilp6 expression represses dilp2 and dilp5 mRNA in the brain, and the secretion of DILP2 into the hemolymph. The longevity benefit of expressing dfoxo in fat body, and the nonautonomous effect of fat body dfoxo upon brain dilp expression, is blocked by simultaneously repressing dilp6 by RNAi in fat body. dilp6 thus appears to bridge dFOXO, adipose tissue and brain endocrine function to regulate Drosophila longevity.

Fig. 1

dilp6 tissue distribution and response to fasting. (A) dilp6 mRNA measured from fat body, midgut, ovary, brain and head carcass. (B) dilp mRNA measured from overnight fasted females: in fat body dilp6 increased upon fasting, in brain, only dilp5 is reduced upon fasting. (C) dfoxo over-expressed (UAS–dfoxo-TM) in fat body (S32: head; S106 abdominal) up-regulates dilp6 mRNA. Asterisk indicates significant difference between treatment and control (P < 0.05). RU: RU486 (mifepristone).

Fig. 2

dilp6 expressed in fat body extends lifespan. (A) dilp6 over-expressed in abdominal fat body (via S106-GS-Gal4) extends lifespan in flies maintained on low-yeast diet, but not (B) when maintained on high-yeast diet. (C) dilp6 over-expressed in head fat body (via S32-GS-Gal4) does not extend lifespan on low-yeast diet but (D) moderately extends lifespan on high-yeast diet. (E) Simultaneous induction of UAS-dfoxo and UAS-dilp6 RNAi inhibits the survival benefit expected from over-expressing dfoxo alone in fat body.

Fig. 3

dilp6 expressed in abdominal fat body modulates metabolism, fecundity and stress resistance. dilp6 over-expressed in abdominal fat body increases fat body (A) triglycerides, (B) glycogen, and (C) trehalose in hemolymph, while (D) repressing fecundity. Asterisk indicates significant difference between treatment and control, P < 0.05. dilp6 over-expressed in abdominal fat body modestly increases the resistance (E) to starvation (log-rank test, P < 0.048, n = 150) and (F) to H₂O₂ (P < 0.0003, n = 130).

Fig. 4

dilp6 expressed in fat body reduces systemic insulin/IGF signaling. 4ebp mRNA is increased in abdominal fat body and thorax when dilp6 is over-expressed in head fat body (A), while 4ebp message is increased in the head and thorax when dilp6 is over-expressed in abdominal fat body (B). dilp2 and dilp5 mRNA measured from the head are reduced when dilp6 is over-expressed in head fat body (C) and abdominal fat body (D). Asterisk indicates significant difference between treatment and control (P < 0.05).

Fig. 5

Fat body dilp6 reduces dilp2 mRNA and dilp2 secretion. (A–F) The cellular distribution of dilp2 (red) and dilp5 (green) changes within IPC when dilp6 is over-expressed in abdominal fat body. (G) Quantified immunostaining fluorescence of dilp2 in the IPC is significantly reduced in dilp6 over-expressing flies. Mean ± SE of eight replicate preparations. (H) EIA assay for dilp peptides in hemolymph. Circulating DILP2 is significantly reduced when dilp6 is over-expressed in abdominal fat body. Asterisk indicates significant difference between treatment and control (P < 0.05). Scale bar: 20 μm.

Fig. 6

dilp6 is a local insulin/IGF agonist and required for non-autonomous effects of dfoxo upon brain insulin/IGF expression. (A) Western blot: phospho-Akt and phospho-FOXO in abdominal fat body are increased when dilp6 is over-expressed in this tissue, quantified in (B; n = 4). (C) 4ebp mRNA in abdominal fat body is reduced when dilp6 was over-expressed in this tissue. Asterisk indicates significant difference between treatment and control (P < 0.05). Simultaneous expression of dfoxo and dilp6(RNAi) in fat body (D) prevents blocks the normal over-expression of dilp6 induced by dfoxo and (E) prevents the repression of dilp2 mRNA at a distance in the brain.