The Attenuation of Insulin/IGF-1 Signaling Pathway Plays a Crucial Role in the Myo-Inositol-Alleviated Aging in Caenorhabditis elegans

Abstract

Myo-Inositol (MI) has been shown to alleviate aging in Caenorhabditis (C). elegans. However, the mechanism by which MI alleviates aging remains unclear. In this study, we investigate whether MI can modulate the PI3K so as to attenuate the insulin/IGF-1 signaling (IIS) pathway and exert the longevity effect. The wild-type C. elegans and two mutants of AKT-1 and DAF-16 were used to explore the mechanism of MI so as to extend the lifespan, as well as to improve the health indexes of pharyngeal pumping and body bend, and an aging marker of autofluorescence in the C. elegans. We confirmed that MI could significantly extend the lifespan of C. elegans. MI also ameliorated the pharyngeal pumping and body bend and decreased autofluorescence. We further adopted the approach to reveal the loss-of-function mutants to find the signaling mechanism of MI. The functions of the lifespan-extending, health-improving, and autofluorescence-decreasing effects of MI disappeared in the AKT-1 and DAF-16 mutants. MI could also induce the nuclear localization of the DAF-16. Importantly, we found that MI could dramatically inhibit the phosphoinositide 3-kinase (PI3K) activity in a dose-dependent manner with an IC50 of 90.2 μM for the p110α isoform of the PI3K and 21.7 μM for the p110β. In addition, the downregulation of the PI3K expression and the inhibition of the AKT phosphorylation by MI was also obtained. All these results demonstrate that MI can inhibit the PI3K activity and downregulate the PI3K expression, and the attenuation of the IIS pathway plays a crucial role for MI in alleviating aging in C. elegans.

Keywords: C. elegans; PI3K inhibitor; insulin/insulin-like growth factor 1 signaling pathway; longevity; myo-inositol.

Figure 1

Effects of the myo-inositol (MI) on the lifespan of wild-type C. elegans. The nematodes (90 worms per group) were grown on plates containing different dosages of MI (0, 25, 50, 100, and 200 nmol/plate) as described in Methods. The results are from a representative experiment out of two independent experiments.

Figure 2

Effects of the myo-inositol (MI) on the pharyngeal pumping, body bends, and autofluorescence of wild-type C. elegans. The nematodes were grown on plates with different dosages of MI (0, 25, 50, 100, and 200 nmol/plate). On the fourth, sixth and eighth days, and the 10th day of life, (a) the pharyngeal pumping (n = 10), (b) body bends (n = 10), and (c) autofluorescence (left: photos; right: quantitative results, n = 3) were determined as described in Methods. For the results of pharyngeal pumping and body bends, the data were analyzed by the simple regression analysis method, but the data of 200 nmol/plate MI were excluded. p values are shown in the figures, or values (mean ± SD) not sharing a common letter are significantly different (p < 0.05). The results are from a representative experiment out of two independent experiments.

Figure 3

Effects of the myo-inositol (MI) on the lifespan and health of the AKT-1 mutants. (a) For the lifespan assay, various mutants (90 worms per group) were grown on plates containing with or without 100 nmol/plate of MI. The surviving worms were counted every two days. The surviving percentages were obtained, and the results were analyzed by the log-rank test with the SPSS software. For the health analysis, the nematodes were grown on plates containing with or without 100 nmol/plate of MI. (b) The pharyngeal pumping (n = 10), (c) body bends (n = 10), and (d) autofluorescence (left: photos; right: quantitative results, n = 3) were determined as described previously. Values are presented as mean ± SD, and p values are shown in the figures. The results are from a representative experiment out of three independent experiments.

Figure 4

Effects of the myo-inositol (MI) on the lifespan and health of the DAF-16 mutants. (a) For the lifespan assay, various mutants (90 worms per group) were grown on plates containing with or without 100 nmol/plate of MI. The surviving worms were counted every two days. The surviving percentages were obtained, and the results were analyzed by the log-rank test with the SPSS software. For the health analysis, the nematodes were grown on plates containing with or without 100 nmol/plate of MI. (b) The pharyngeal pumping (n = 10), (c) body bends (n = 10), and (d) autofluorescence (left: photos; right: quantitative results, n = 3) were determined as described previously. Values are presented as mean ± SD, and p values are shown in the figures. The results are from a representative experiment out of three independent experiments.

Figure 5

Effects the myo-inositol (MI) on the DAF-16 nuclear localization. After the MQD1543 strain of nematodes was grown on plates with different dosages of MI (0, 50 and 100 nmol/plate) for five days, the DAF-16:GFP fluorescence was measured as described in Methods. The representative photos at 200× magnification of (a) cytosolic localization, (b) intermediate localization, (c) nuclear localization, and (d) the semi-quantitative results of the DAF-16 distribution are shown. The data are from three independent experiments using 30 nematodes per group.

Figure 5

Effects the myo-inositol (MI) on the DAF-16 nuclear localization. After the MQD1543 strain of nematodes was grown on plates with different dosages of MI (0, 50 and 100 nmol/plate) for five days, the DAF-16:GFP fluorescence was measured as described in Methods. The representative photos at 200× magnification of (a) cytosolic localization, (b) intermediate localization, (c) nuclear localization, and (d) the semi-quantitative results of the DAF-16 distribution are shown. The data are from three independent experiments using 30 nematodes per group.

Figure 6

Effects of the myo-inositol (MI) on the expressions of PI3K, AKT, and PTEN, and the phosphorylation of AKT in Hs68 cells. After being treated with various concentrations of MI and LY294002 for five days, the cell lysates were obtained. The protein expressions or AKT phosphorylation level in the cell lysates were detected by the Western blot method as described in Methods. (a) Western blot Images; (b) Western blot quantification results (n = 3 independent experiments). Values (mean ± SD) not sharing a common letter are significantly different (p < 0.05).

Figure 7

Effects of the myo-inositol (MI) on the phosphoinositide 3-kinase (PI3K) activity. Two isoforms of the PI3K enzyme, (a) p110α and (b) p110β, were incubated with different concentrations of MI. The PI3K activity was detected by a commercial PI3K kit as described in Methods. The inhibition percentage of MI on the PI3K isozymes is expressed as the mean ± SD (n = 3 independent experiments); Values not sharing a common letter are significantly different (p < 0.05).

Figure 8

Effects of MI on the PTEN activity in vitro. The PTEN enzyme was incubated with different concentrations of MI (0–1000 μM). The PTEN activity was detected by a commercial Malachite Green Assay Kit as described in Methods. The relative PTEN activities are expressed as the mean ± SD (n = 3 independent experiments); Values not sharing a common letter are significantly different (p < 0.05).

Figure 9

The schematic presentation of the mechanisms by which MI inhibits the signaling of insulin/insulin-like growth factor 1 (IIS) pathway. The symbol as: “The inhibition step of MI or the inactivation step in the signaling pathway”, the symbol ⇩ as “The detected downregulation of protein expressions or protein phosphorylation by MI”, and ⇧ as “The detected upregulation of protein expression by MI” in this study. NB: the references are indicated in square brackets.