Activation of the TOR Signalling Pathway by Glutamine Regulates Insect Fecundity

Abstract

The target of rapamycin (TOR) positively controls cell growth in response to nutrients such as amino acids. However, research on the specific nutrients sensed by TOR is limited. Glutamine (Gln), a particularly important amino acid involved in metabolism in organisms, is synthesised and catalysed exclusively by glutamine synthetase (GS), and our previous studies have shown that Gln may regulate fecundity in vivo levels of the brown planthopper (BPH) Nilaparvata lugens. Until now, it has remained unclear whether Gln activates or inhibits the TOR signalling pathway. Here, we performed the combined analyses of iTRAQ (isobaric tags for relative and absolute quantification) and DGE (tag-based digital gene expression) data in N. lugens at the protein and transcript levels after GS RNAi, and we found that 52 pathways overlap, including the TOR pathway. We further experimentally demonstrate that Gln activates the TOR pathway by promoting the serine/threonine protein kinase AKT and inhibiting the 5'AMP-activated protein kinase AMPK phosphorylation activity in the pest. Furthermore, TOR regulates the fecundity of N. lugens probably by mediating vitellogenin (Vg) expression. This work is the first report that Gln activates the TOR pathway in vivo.

Figure 1. Combined analyses of the iTRAQ and DGE data after GS RNAi.

First day brachypterous female adults were injected with dsGS or dsGFP. Samples were used for DGE and iTRAQ 48 h and 72 h post-injection, respectively. (A) No. of differentially expressed proteins identified by iTRAQ, the conditions for protein spots were ≥1.2-fold or ≤0.8 (p < 0.05) for up-regulated or down-regulated proteins in dsGS compared to dsGFP. (B) The representative MS/MS spectra of down-regulated proteins, GS (NLU012724.1) and Vg (NLU019204.1). (C) No. of differentially expressed unigenes identified by DGE, the conditions for unigenes was FDR ≤0.001 and |log₂Ratio| ≥ 1. (D) GO analysis of differentially expressed genes. (E) Venn diagrams of differentially expressed genes/proteins from iTRAQ and DGE analyses. (F) Cluster of pathways for iTRAQ and DGE. (G) Venn diagrams of regulated pathways from iTRAQ and DGE analyses.

Figure 2. Effect of GS knockdown on gene expression and enzyme activity.

First day brachypterous female adults were injected with 50 nL dsGS (5 ng/nL), dsGFP (5 ng/nL), ddH₂O or MSX (10 mM), respectively. (A) The transcript levels of genes after injection by qRT-PCR. Data represent mean values±S.E.M (n = 3), *p < 0.05, **p < 0.01. (B) The relative activity of GS at 48 h post-injection. Data represent mean values±S.E.M (n = 3), and those in the columns followed by the different letters mean significant difference (p = 0.05, Duncan’s multiple range test). Inset shows Western blotting analysis of GS protein levels injected with either dsGFP or dsGS; the β-actin gene was used as an internal control. (C) The transcript levels of Vg at 48 h post-injection by qRT-PCR. Data represent mean values±S.E.M (n = 3), and those in the columns followed by the different letters mean significant difference (p = 0.05, Duncan’s multiple range test). Inset shows Western blotting analysis of Vg protein levels injected with either dsGFP or dsGS; the β-actin gene was used as an internal control.

Figure 3. Western blots showing S6 K phosphorylation levels and GS and Vg protein abundance under various nutritional conditions at 48 h post-injection.

(A) First day brachypterous female adults were injected with 50 nL dsGFP (5 ng/nL), dsGS, dsTOR, MSX (10 mM), Gln (20 mM) and ddH₂O. Shown are representative blots (n = 3). (B) The 3^rd to the 5^th instar nymphs were fed an artificial diet (w/w Gln) together with dsGS or dsGFP (0.5 μg/μl). Shown are representative blots (n = 3).

Figure 4. The effect of TOR on fecundity in N. lugens.

First day brachypterous female adults were injected with dsTOR or dsGFP. (A) The transcript levels of TOR after injection with dsRNA. (B) The transcript levels of Vg after RNAi. Data represent the mean values ± S.E.M of three replicates, ‘*’ indicates statistically significant difference (t-test, p < 0.05). The inset shows Western blotting analysis of Vg protein levels, and the β-actin gene was used as an internal control. (C) The frequency distribution of N. lugens offspring.

Figure 5. Proposed mechanism of glutamine-mediated activation of the TOR signalling pathway

. (A) The relative activity of AKT at 48 h post-injection. Data represent the mean values±S.E.M (n = 3), and the values in the columns followed by different letters note a significant difference (p = 0.05, Duncan’s multiple range test). (B) Western blots showing S6 K phosphorylation levels and GS protein abundance. Shown are representative blots (n = 3). (C) The relative activity of AMPK at 48 h post-injection. Data represent the mean values±S.E.M (n = 3), and the values in the columns followed by the different letters note a significant difference (p = 0.05, Duncan’s multiple range test). (D) Western blots showing S6K phosphorylation levels and GS protein abundance. Shown are representative blots (n = 4). (E) The mRNA expression levels at 48 h after injection of dsAKT. Data represent the mean values±S.E.M (n = 3), *p < 0.05, **p < 0.01. (F) The relative activity of GS at 48 h after injection of dsAKT. Data represent the mean values±S.E.M (n = 3), *p < 0.05. (G) Proposed model of the activation of the TOR signalling pathway by Gln by promoting AKT and inhibiting AMPK in N. lugens.