Long-lived Indy and calorie restriction interact to extend life span

Abstract

Calorie restriction (CR) improves health and extends life span in a variety of species. Despite many downstream molecules and physiological systems having been identified as being regulated by CR, the mechanism by which CR extends life span remains unclear. The Drosophila gene Indy (for I'm not dead yet), involved in the transport and storage of Krebs cycle intermediates in tissues important in fly metabolism, was proposed to regulate life span via an effect on metabolism that could overlap with CR. In this study, we report that CR down regulates Indy mRNA expression, and that CR and the level of Indy expression interact to affect longevity. Optimal life span extension is seen when Indy expression is decreased between 25 and 75% of normal. Indy long-lived flies show several phenotypes that are shared by long-lived CR flies, including decreased insulin-like signaling, lipid storage, weight gain, and resistance to starvation as well as an increase in spontaneous physical activity. We conclude that Indy and CR interact to affect longevity and that a decrease in Indy may induce a CR-like status that confers life span extension.

Fig. 1.

The interaction between CR and Indy affects life span. (A) Median life span of yw control flies (+/+, blue line), Indy206 heterozygous (+/206, red line), and Indy206 homozygous (206/206, green line) flies on 5% (0.5 N), 10% (1 N), and 15% (1.5 N) dextrose and yeast diets. (B) Diets regulate Indy mRNA expression in +/+ and +/206 flies. Data are presented as mean ± SD. Experiments were done in triplicate and each sample contained more than 20 heads and thoraxes of 20-day-old male flies. The * indicates P < 0.05 by t-test. (C) A heat map presentation showing correlations between diets, Indy mRNA expression and median lifespan of flies. Data were derived from A and B. ●, (+/+); ■, (+/206); ▴, (206/206).

Fig. 2.

CR and Indy alter insulin-like signaling. (A) Real-time PCR detection of mRNA expression levels of dilp2, dilp3, and dilp5 in yw control flies (+/+) on high-calorie food (1.5 N), yw control flies (+/+) on low-calorie food (0.5 N/CR), and Indy206 heterozygous (+/206) flies on high-calorie food (1.5 N). Data are presented as mean ± SD. Experiments were done in triplicate and each sample contained more than 20 heads and thoraxes of 20-day-old male flies. The * indicates P < 0.05 by t-test. (B–D) Immunohistochemical localization of dFOXO in the abdominal fat body of yw control (B and C) or Indy206 heterozygous (D) flies on high-calorie (1.5 N) food (B and D) or low-calorie (0.5 N/CR) food (C). Sections were visualized using a red fluorescent-conjugated secondary antibody for dFOXO and counter stained with DAPI for blue nuclear fluorescence. The white arrows indicate co-localization of dFOXO and DAPI in nuclei (pink). (Scale bar, 50 μm.)

Fig. 3.

CR and Indy induce reduced starvation resistance and body weight gain. (A) Twenty-day-old yw control flies (+/+) on high-calorie food (1.5 N, blue line) or low-calorie food (0.5 N/CR, red line), and Indy206 heterozygous flies (+/206) on high-calorie food (1.5 N, green line) were subjected to starvation challenge. Survivorship curves were collected from more than 100 flies and presented as mean ± SEM. Similar results were obtained from 2 other replicate experiments. (B) Total triglyceride measurements of flies on food conditions described above. Data are presented as mean ± SD. Experiments were done in triplicate and each sample contained more than 10 flies. The * indicates P < 0.05 by t test. (C) Change of body weight over a 20-day period was measured in flies on food conditions described above. Data are presented as mean ± SD. Experiments were done in triplicate, and more than 100 flies in each group were measured. (D–K) Oil Red O staining of yw control flies on 1.5 N food (D and H), 16-h fasted yw control flies on 1.5 N food (E and I), 16-h fasted yw control flies on 0.5 N/CR food (F and J), and 16-h fasted Indy heterozygous flies on 1.5 N food (G and K). Blue color is hemotoxylin counter stain. [Scale bars, 500 μm (D–G) and 250 μm (H–K).]

Fig. 4.

Indy and CR induce increased physical activity. Activities of 20-day-old yw control (+/+) flies on 1.5 N food (blue) or 0.5 N/CR food (red) and Indy heterozygous (+/206) flies on 1.5 N food (green) were recorded over a 48-h period using Drosophila activity monitor. Data were collected every 20 s and presented as total movement of 10 flies. Experiments were done in triplicate.