The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate

Abstract

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. Here we found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, our study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding our understanding of the comprehensive regulatory mechanisms of final body size in animals.

Keywords: 20-hydroxyecdysone; AMPK-PP2A axis; fat body; growth rate; insulin/IGF signaling.

Fig. 1.

AMPK inhibits IIS in Bombyx. (A) The developmental phosphorylation profile of AMPK, InR, and 4EBP in the Bombyx fat body from the early fourth instar to the prepupal stage. (B) The developmental profiles of mRNA levels of AMPKα (blue), AMPKβ (red), and AMPKγ (green) in the Bombyx fat body as well as ecdysteroid titers (71) from the early fourth instar to the prepupal stage. Fold-changes shown are relative to Rp49. (C and D) Metformin or AICAR treatment activated AMPK and reduced the phosphorylation of InR and 4EBP. AC, the treatment of AICAR; Ctrl, control. (E) Compound C treatment reduced the phosphorylation of AMPK and promoted InR and 4EBP. CC, the treatment of compound C. (F) Phosphorylation levels of the target proteins when the Bombyx cell line DZNU-Bm-12 treated with AICAR, compound C, or both. (G) The phosphorylation levels of the target proteins when the active form of AMPK (V5-AMPK^CA) overexpressed constitutively in DZNU-Bm-12 cells. Note: Overexpression of EGFP was used as control.

Fig. 2.

AMPK inhibits IIS in Drosophila. (A) Developmental phosphorylation profile of AMPK, AKT, InR, S6K, and 4EBP in the Drosophila fat body at different stages (feeding, EW, LW, WPP, and 6 h after pupation). (B) Developmental profiles of mRNA levels of AMPKα (blue), AMPKβ (red), and AMPKγ (green) in the Drosophila fat body and ecdysteroid titers (72, 73) at different stages. Fold-changes shown are relative to Rp49. (C) Phosphorylation levels of InR, AKT, S6K, and 4EBP were decreased in Adh-Gal4 > UAS-AMPK^CA. Adh-Gal4 drives fat body-specific Gal4 expression. (D) Phosphorylation levels of InR, AKT, S6K, and 4EBP were increased in Lsp2-Gal4 > UAS-AMPK^DN. Lsp2-Gal4 also drives fat body-specific Gal4 expression. (E) The Flp-out experiment revealing that the activity of PI3K is inhibited in red-positive clones in HsFlpase; Act > CD2 > Gal4, UAS-RFP, tGPH; UAS-AMPK^CA at the feeding stage. RFP (red), tGPH (green). (F) The Flp-out experiment revealing that the activity of PI3K is increased in red-positive clones in HsFlpase; Act > CD2 > Gal4, UAS-RFP, tGPH::UAS-AMPK^DN at the LW stage. RFP (red), tGPH (green).

Fig. 3.

AMPK acts through PP2A to inhibit IIS in Drosophila fat body. (A, Upper) Developmental profile of the relative PP2A activity in the Drosophila fat body at different stages (feeding, EW, LW, WPP, and 6 h after pupation). Fold-changes shown are relative to the feeding stage; (Lower) Relative PP2A activities in the fat body of different Drosophila genotypes (W¹¹¹⁸, Lsp2-Gal4 > UAS-AMPK^DN, Lsp2-Gal4 > UAS-PP2A^CA, Lsp2-Gal4 > UAS-AMPK^DN::UAS-PP2A^CA, Lsp2-Gal4 > UAS-AMPK^CA, Lsp2-Gal4 > UAS-PP2A^DN, and Lsp2-Gal4 > UAS-PP2A^DN; UAS-AMPK^CA). Fold-changes shown are relative to the W¹¹¹⁸. Statistical significance between samples was evaluated using ANOVA: Bars labeled with different lowercase letters are significantly different (P < 0.05). (B) Western blot analysis results in the fat body of different Drosophila genotypes as in A, Lower. (C) A Flp-out experiment revealing that the activity of PI3K is decreased in red-positive clones in HsFlpase; Act > y⁺ > Gal4, UAS-RFP, tGPH::UAS-PP2A^CA and HsFlpase; Act > y⁺ > Gal4, UAS-RFP, UAS-AMPK^DN::UAS-PP2A^CA, tGPH at the feeding stage, while the activity of PI3K is increased in HsFlpase; Act > y⁺ > Gal4, UAS-RFP, UAS-PP2A^DN, and HsFlpase; Act > y⁺ > Gal4, UAS-RFP, UAS-PP2A^DN::tGPH; UAS-AMPK^CA at the LW stage. RFP (red), tGPH (green).

Fig. 4.

20E activates the AMPK-PP2A axis and inhibits IIS in insect fat body. (A) p-AMPKα, total-AMPKα, p-InR, and p-4EBP levels were measured after the 20E treatment (5 μg per larva) or USP RNAi in Bombyx. (A, Right) Anti-USP was used. (B) The levels of p-AMPKα, total-AMPKα, p-InR, p-AKT, p-S6K, and p-4EBP were measured in 20E-fed (2.5 μg/μL) or Lsp2 > EcR^DN Drosophila. (C) tGPH membrane localization in the fat body of Drosophila following different treatments (5% DMSO and 2.5 μg/μL 20E). tGPH (green). (D) Relative PP2A activity in 20E-fed (2.5 μg/μL) or Lsp2 > EcR^DN Drosophila. Fold-changes shown are relative to the W¹¹¹⁸. Statistical significance between samples was evaluated using ANOVA: Bars labeled with different lowercase letters are significantly different (P < 0.05). (E) Flp-out experiment revealing that the activity of PI3K is increased in red-positive clones in HsFlpase; Act > CD2 > Gal4, UAS-RFP, tGPH::UAS-EcR^DN at the LW stage. RFP (red), tGPH (green). (F and G) mRNA levels of AMPKα, AMPKβ, and AMPKγ in the fat body of Bombyx following 20E treatment (6 h and 12 h) or USP RNAi. Fold-changes shown are relative to DMSO treatment or GFP RNAi, respectively. (H and I) mRNA levels of AMPKα, AMPKβ, and AMPKγ in the fat body of Drosophila 6 h after the 20E treatment or Lsp2 > EcR^DN. Fold-changes shown are relative to DMSO treatment or Lsp2 > W¹¹¹⁸, respectively. Statistical significance between samples was evaluated using Student’s t test (*P < 0.05, **P < 0.01).

Fig. 5.

The AMPK-PP2A axis is required for 20E to antagonize IIS in insect fat body. (A) p-AMPKα, total-AMPKα, p-InR, and p-4EBP levels were measured in Bombyx following treatment with 20E and compound C. CC, compound C; CK, control. (B and C) Flp-out experiment revealing that the activity of PI3K is increased in red-positive clones in HsFlpase; Act > y⁺ > Gal4, UAS-RFP, tGPH::UAS-AMPK^DN (B) or HsFlpase; Act > y⁺ > Gal4, UAS-RFP, tGPH::UAS-PP2A^DN (C) following 20E treatment (2.5 μg/μL) at the LW stage. RFP (red), tGPH (green).

Fig. 6.

Activation of AMPK-PP2A axis and inhibition of IIS in insect fat body reduce food consumption and restrict growth rate. (A–D) Changes in lethality (A), phenotype (B), body weight (C), and percentage of pupariation (D) following the metformin treatment of different dosages in Bombyx on L5D2. (E–H) Changes in percentage of pupariation (E), pupa size (F), body weight (G), and food consumption (H) in ppl > W¹¹¹⁸ (control), ppl > AMPK^CA, ppl > PP2A^CA, ppl > PTEN, and ppl > Dp110^DN. *P < 0.05, **P < 0.01, ***P < 0.001.