Figure 1.
The signaling network involved in PTTH-stimulated ecdysteroidogenesis in prothoracic gland cells.
Solid lines indicate demonstrated or highly likely relations; dashed lines indicate hypothetical interactions; dotted lines indicate the relationship demonstrated in the present study. PTTH, prothoracicotropic hormone; Ras, Ras GTP-binding protein; ERK, extracellular signal-regulated kinase; cAMP, cyclic adenosine monophosphate; PI3K, phosphatidylinositol 3-kinase; TOR, target of rapamycin; 4E-BP, eIF4E-binding protein; S6K, p70 ribosomal protein S6 kinase; AMPK, adenosine 5′-monophosphate-activated protein kinase (ref. 2, 3 and this work).
Figure 2.
Comparison of predicted N-terminal amino acid sequences of the phosphorylation domains of Bombyx AMPK with their counterparts from Drosophila, mouse, and human.
The plus sign (+) indicates a conserved amino acid residue (Thr) that is phosphorylated. Asterisks (*) indicate identical amino acids. AMPKs of various species were cited from the NCBI, and amino acid sequences were registered with the following accession numbers: ABQ62953.1 (Bombyx), AF020310.1 (Drosophila), AAB95475.1 (mouse), and AAB32732.1 (human).
Figure 3.
Effects of PTTH (A), AICAR (B), compound C (C), and lambda protein phosphatase (D) on AMPK phosphorylation.
Con, glands incubated in control medium; P, glands incubated in medium containing PTTH; Al, glands incubated in medium containing 1 mM AICAR; In, glands incubated in medium containing 10 µM compound C; Bu, glands treated with buffer only; Lp, glands treated with lambda protein phosphatase. Results shown are representative of two independent experiments.
Figure 4.
Time- and dose-dependent inhibitory effects of AMPK phosphorylation by PTTH.
Prothoracic glands were either treated with PTTH for the indicated time points (A), treated with indicated concentrations of PTTH, or incubated with control medium (con) for 60 min (B). Results shown are representative of three independent experiments.
Figure 5.
In vivo inhibition of AMPK phosphorylation of prothoracic glands by PTTH.
P, larvae injected with saline containing PTTH; C, larvae injected with saline only. Results shown are representative of three independent experiments.
Figure 6.
Effects of LY294002 (A) and U0126 (B) onPTTH-inhibited AMPK phosphorylation.
C, glands incubated in control medium; P, glands incubated in medium containing PTTH only; L, glands incubated in medium containing 50 µM LY294002 only; P+L, glands incubated in medium containing both PTTH and LY294002; U, glands incubated in medium containing 10 µM U0126 only; P+U, glands incubated in medium containing both PTTH and U0126. Results shown are representative of three independent experiments.
Figure 7.
Effects of AICAR on PTTH-regulated AMPK phosphorylation (A) and ecdysteroidogenesis (B).
C, glands incubated in control medium; P, glands incubated in medium containing PTTH only; A, glands incubated in medium containing 1 mM AICAR only; P+A, glands incubated in medium containing both PTTH and AICAR. Results shown for the Western blot analysis are representative of three independent experiments. Each bar represents the mean ± SEM (N = 8). Different letters above the bars indicate a significant difference (ANOVA followed by Tukey’s multiple-comparisons test, p<0.01).
Figure 8.
Effects of AICAR on PTTH-stimulated TOR signaling.
C, Glands incubated in control medium; P, glands incubated in medium containing PTTH only; A, glands incubated in medium containing 1 mM AICAR only; P+A, glands incubated in medium containing both PTTH and AICAR. Results shown are representative of three independent experiments.
Figure 9.
Effects of AICAR on PTTH-stimulated ERK phosphorylation.
C, Glands incubated in control medium; P, glands incubated in medium containing PTTH only; A, glands incubated in medium containing 1 mM AICAR only; P+A, glands incubated in medium containing both PTTH and AICAR. Results shown are representative of three independent experiments.
Figure 10.
Changes in mRNA expression levels of AMPKα (A), β (B), and γ (C) upon treatment with PTTH.
Prothoracic glands from day 7 last instar larvae were preincubated in medium for 30 min, then transferred to medium containing PTTH (P) or control medium (C). The incubation was maintained for 1, 2, and 8 h. After incubation, total RNA was extracted from the prothoracic glands, and mRNA expression levels of AMPK were determined by a qRT-PCR. Each bar represents the mean ± SEM of three separate assays.