Physiological effects of manipulating the level of insulin-degrading enzyme in insulin-producing cells of Drosophila

Abstract

Insulin-degrading enzyme (IDE) degrades insulin and other peptides, including the Aβ peptide of Alzheimer's disease. However, the mechanism by which IDE acts on its substrates in vivo is unclear, and its role in pathogenesis of type 2 diabetes and Alzheimer's disease is controversial. Here, we show that in Drosophila knocking down IDE in insulin-producing cells (IPCs) of the brain results in increased body weight and fecundity, decreased circulating sugar levels, and reduced lifespan. Moreover, knocking down and over-expressing IDE in IPCs have opposite physiological effects. As mis-regulated insulin signaling in peripheral tissues is known to cause similar phenotypes, our data suggest a role for Drosophila IDE in determining the level of insulin-like peptides made by IPCs that systemically activate insulin signaling.

Figure 1

IDE is important for body weight. (A) The broadly expressed T80-Gal4 driver was used to activate Ide RNAi in virtually all tissues during larval development. Quantitative RT-PCR analysis, using the same amount of total RNA isolated from 3^rd instar larvae of the same age, indicated that Ide mRNA was reduced by 77–87% in three independent Ide RNAi lines (T80G4/IDE^RNAi) compared to the control where the driver was crossed into the w¹¹¹⁸ background lacking the Ide RNAi construct (T80G4/+). (B) The broadly expressed act-Gal4 driver or the IPC-specific dilp2-Gal4 driver was used to activate Ide RNAi. The body weight increased by 25% in actG4/IDE^RNAi flies (n = 195) and by 21% in dilp2G4/IDE^RNAi flies (n = 134) when compared to the respective controls (actG4/+, n = 188; dilp2G4/+, n = 134). 7 day-old adult females were weighed. (C) Flies in which Ide expression is broadly activated (actG4/dIDE, n = 618) weighed 11% less than control flies (actG4/+, n = 878). Seven-day-old adult females were weighed. *p < 0.0002, two-tailed t-test. (D) IDE overexpressing flies (actG4/dIDE) were generally smaller than control flies (actG4/+). Flies of the two different genotypes were cultured separately but in parallel under identical conditions as possible to avoid over-crowding. The flies shown in this figure were randomly selected from culture vials and photographed about 1 day after eclosion.

Figure 2

IDE is important for circulating sugar level and fecundity. (A) Hemolymph total glucose, derived mostly from trehalose and representing ≤1% free glucose, was about 30% lower for flies in which Ide RNAi was activated with the dilp2-Gal4 driver (dilp2G4/IDE^RNAi) when compared to the control (dilp2G4/+). *p = 0.0127, one-tailed t-test. The circulating sugar level was not determined for IDE overexpressing flies. (B) Using the dilp2-Gal4 driver to activate Ide RNAi (dilp2G4/IDE^RNAi) or IDE overexpression (dilp2G4/dIDE) in IPCs resulted in flies with increased and decreased fecundity, respectively, compared to control flies (dilp2G4/+). Eggs laid by individual virgin females were counted, with a plot showing the average of data from 50–60 females per genotype minus any flies that died during the experiment. The dilp2G4/+, dilp2G4/IDE^RNAi and dilp2G4/dIDE females, respectively, laid per female an average of about 1,090, 1,250 and 970 eggs over 39 days, or about 28, 32 and 25 eggs per day. Preliminary experiments with mated females showed similar results.

Figure 3

IDE is important for lifespan. (A) Flies in which IDE was knocked down in IPCs with the dilp2-Gal4 driver (dilp2G4/IDE^RNAi, n = 381) had 18% lower median lifespan compared to control flies carrying the driver alone (dilp2G4/+, n = 383). Results are shown for female flies cultured at 29°C to enhance knockdown efficiency. Similar results were obtained with male flies. (B) Flies in which Drosophila IDE was overexpressed in IPCs with the dilp2-Gal4 driver (dilp2G4/dIDE, n = 328) had 14% greater median lifespan compared to control flies carrying the driver alone (dilp2G4/+, n = 288). Results are shown for female flies cultured at 25°C. (C) Flies in which human IDE was overexpressed in IPCs with the dilp2-Gal4 driver (dilp2G4/hIDE, n = 374) had 18% greater median lifespan compared to control flies carrying the driver alone (dilp2G4/+, n = 344). Results are shown for female flies cultured at 25°C.