JH and ILP2 regulate carbohydrate, protein, and lipid metabolism during starvation.

<p>A. Total carbohydrate, protein, and lipid levels were determined by Anthrone reagent, Bradford, and vanillin reagent respectively in samples collected from day 3 to day 8 starved and fed male beetles. Three beetles were used for each time point and six biological replicate were used. The Means+S.D (n = 6) are shown. B. The nutrient levels including carbohydrate, protein, and lipid are regulated by JH and ILP2 during starvation. The male beetles injected with <i>malE</i>, ILP2, or JHAMT dsRNA were collected on day 8. The total carbohydrate, protein, and lipid were determined. Shown are the Means+S.D (n = 6). Asterisks show treatments that are significantly different (<i>P</i><0.05) by one-way ANOVA.</p

JH and insulin regulate starvation resistance.

<p>A. RNAi-aided knockdown in the expression of genes coding for JHAMT or Met extended the starvation survival. Percent beetles survived during starvation after injection of control <i>malE</i>, JHAMT, or Met dsRNA respectively into 40 newly emerged male adults are shown. Starvation survival was recorded from day 4 to day 14. B. RNAi-aided knockdown in the expression of genes coding for ILP2 extend starvation survival. Percent beetles survived during starvation after injection of control <i>malE</i>, ILP1, ILP2, ILP3, or ILP4 dsRNA respectively into 40 new emerged male adults are shown. Starvation survival was recorded from day 4 to day 14. C. Bovine insulin can rescue the starvation survival of JHAMT or ILP2 RNAi beetles. Percent beetles survived after injection of control <i>malE</i>, JHAMT, ILP2 dsRNA into day 0 male adults followed by injection of either 25 mM HEPES or 25 mM HEPES containing 10 mg/ml insulin into day 5 adults are shown. The starvation survival was recorded from day 9 to day 14. D. JH III rescues starvation survival in JHAMT but not in ILP2 RNAi beetles. Shown are percentages of beetles survived after injection of control <i>malE</i>, JHAMT, ILP2 dsRNA into day 0 male adults followed by topical application of either acetone or 10 mM JH III in acetone on days 3, 5, and 7. The starvation survival was recorded from day 9 to day 14.</p

The relative mRNA levels of TRET and trehalase in the alimentary canal, fat body, and head after manipulation of JH or insulin levels.

<p>A. The relative mRNA levels of TRET in the alimentary canal, fat body, and head after injection of <i>malE</i>, JHAMT, ILP2, or Met dsRNA into newly emerged male adults. Total RNA was isolated and used to measure relative TRET mRNA by qRT-PCR using RP49 as a control. The data shown are the Mean+S.D. (n = 3). B. The relative mRNA levels of TRET in the alimentary canal, fat body, and head after topical application of 0.5 µl acetone, 0.5 µl 10 mM JH III in acetone, or injection of 0.3 µl HEPES solution or bovine insulin in HEPES solution at 10 mg/ml concentration. Total RNA was isolated from the tissues including fat body, alimentary canal, and head dissected from the starved beetles at 6 hours after treatment on day 5 for JH and insulin induction experiment. The total RNA was converted to cDNA, and the relative TRET mRNA levels were determined by qRT-PCR using RP49 as a control. The data shown are the Mean+S.D. (n = 3). C. Same treatments as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003535#pgen-1003535-g005" target="_blank">Figure 5A</a> except trehalose mRNA levels were determined. D. Same treatments as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003535#pgen-1003535-g005" target="_blank">Figure 5B</a> except trehalose mRNA levels were determined.</p

Juvenile Hormone and Insulin Regulate Trehalose Homeostasis in the Red Flour Beetle, <i>Tribolium castaneum</i>

<div><p>Insulin/IGF-1 signaling (IIS) has been well studied for its role in the control of life span extension and resistance to a variety of stresses. The <i>Drosophila melanogaster</i> insulin-like receptor (InR) mutant showed extended life span due to reduced juvenile hormone (JH) levels. However, little is known about the mechanism of cross talk between IIS and JH in regulation of life span extension and resistance to starvation. In the current study, we investigated the role of IIS and JH signaling in regulation of resistance to starvation. Reduction in JH biosynthesis, JH action, or insulin-like peptide 2 (ILP2) syntheses by RNA interference (RNAi)-aided knockdown in the expression of genes coding for juvenile hormone acid methyltransferase (JHAMT), methoprene-tolerant (Met), or ILP2 respectively decreased lipid and carbohydrate metabolism and extended the survival of starved beetles. Interestingly, the extension of life span could be restored by injection of bovine insulin into JHAMT RNAi beetles but not by application of JH III to ILP2 RNAi beetles. These data suggest that JH controls starvation resistance by regulating synthesis of ILP2. More importantly, JH regulates trehalose homeostasis, including trehalose transport and metabolism, and controls utilization of stored nutrients in starved adults.</p></div

Trehalose metabolism plays an important role in extending life span during starvation.

<p>A. Starvation survival after feeding 10% trehalose plus cellulose, 10% glucose plus cellulose, or cellulose alone to the 8-day-old starved male beetles. The survival was recorded from day 9 to day 12. B. The ratio between hemolymph glucose and trehalose on days 4, 5, and 6 after injection of <i>malE</i>, ILP2, JHAMT, Met, TRET, or TPS dsRNA into the newly emerged adults. The hemolymph was extracted from three beetles for each treatment. The trehalose concentrations were determined using the glucose reagent and trehalase. The data shown are the Mean+S.D. (n = 6). C. Starvation survival after manipulation of endogenous trehalose level by injecting TRET, TPS, or trehalase dsRNA on day 0. The beetles were starved until 14 days. The survival was recorded from day 9 to day 14. D. The relative mRNA level of TPS, TRET, and trehalase after injecting <i>malE</i>, JHAMT, or ILP2 dsRNA. Total RNA was isolated on day 5 from beetles injected with control <i>malE</i>, JHAMT, or ILP2 dsRNA and starved. The RNA was converted to cDNA, and the relative levels of TPS, TRET, and trehalase mRNA were determined by qRT-PCR using RP49 as a control. The data shown are the Mean+S.D. (n = 3). Asterisks show treatments that are significantly different from the control (<i>P</i><0.05) in one-way ANOVA.</p

The relative mRNA levels of TRET, TPS, and trehalase in the fat body, head, male accessory gland, testis, and alimentary canal.

<p>A. Total RNA was isolated from the tissues dissected from the fed beetles from day 1 to day 4, the total RNA was converted to cDNA, and the relative TPS, TRET, and trehalase mRNA levels were determined by qRT-PCR using RP49 as a control. The data shown are the Mean+S.D. (n = 3). Asterisks show treatments that are significantly different from the control (<i>P</i><0.05) in one-way ANOVA. B. The relative mRNA level of TRET, TPS, and trehalase in the starved and fed male beetles collected on day 1 to day 5 after adult emergence. Total RNA was isolated from the whole body of both fed and starved beetles on days 1 to 5. The total RNA was converted to cDNA, and the relative TPS, TRET, and trehalase mRNA levels were determined by qRT-PCR using RP49 as a control. The data shown are the Mean+S.D. (n = 3).</p

MET Is Required for the Maximal Action of 20-Hydroxyecdysone during <em>Bombyx</em> Metamorphosis

<div><p>Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone (20E; the molting hormone) action. Here we report that two MET proteins found in the silkworm, <em>Bombyx mori</em>, participate in 20E signal transduction. <em>Met</em> is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of <em>EcR</em>-<em>USP</em> (the ecdysone receptor genes), RNAi knockdown of <em>Met</em> at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling (including autophagy, apoptosis and destruction of larval tissues and generation of adult structures) and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during <em>Bombyx</em> metamorphosis.</p> </div

Lethal and defective phenotypes caused by <i>Met</i> RNAi in silkworms.

<p>dsRNA (10 µg per larva) was injected into selected larva during initiation of the early wandering stage. <i>egfp</i> dsRNA was used as a control. (A–C) Typical <i>Met1</i> RNAi and <i>Met2</i> RNAi treated silkworms died during the wandering stage (A) or during pupation (B), while some were arrested at the mid-pupal stage (C). The pictures (A–C) show the dying animals after <i>Met</i> RNAi. (D, E) <i>Met</i> RNAi affected adult structure formation. The surviving <i>Met1</i> RNAi and <i>Met2</i> RNAi treated pupae did not fully develop legs and wings during the late pupal stage (D). Many of the surviving <i>Met1</i> RNAi adults failed to shed the pupal cuticle attached to their head or abdomen, exhibiting shortened and distorted legs or unexpanded wings (E).</p

Physical interaction between MET and EcR-USP.

<p>(A) The CytoTrap yeast two-hybrid analyses revealed direct associations among MET1, MET2, EcR and USP. Strong associations between bait and prey proteins led to more yeast colonies. (B) When the <i>HA-EcR</i>, <i>FLAG-USP</i>, and <i>V5-Met1</i> constructs were co-transfected into human HEK 293 cells, 20E treatment for 6 hr at a final concentration of 1 µM had little or no stimulating effects on the physical interactions between MET and EcR-USP. In the immunoprecipation experiments, the bottom Western blot is input. IP, immunoprecipitate; Blot, Western blot. (C) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into the human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated on a 5% native PAGE gel followed by EMSA. Addition of the HA or FLAG antibody resulted in a shift of EcRE. In (C) and (D), the shift was indicated by a black arrow in comparison with a gray arrow. (D) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated 5% native PAGE followed by EMSA. When the V5 or cMyc antibody was added, binding of EcR-USP-EcRE was shifted by MET1 and MET2 in EMSA showing that MET, EcR-USP and EcRE form a protein-DNA complex. (E) <i>Met1</i> RNAi and transfection were simultaneously conducted in <i>Bombyx</i> DZNU-Bm-12 cells for 48 hr, followed by 20E treatment for 6 hr at a final concentration of 1 µM, and measurements of EcRE-driven luciferase activity were done. MET is required for 20E function to induce gene expression via the ecdysone receptor and EcRE. The bars labeled with different lowercase letters are significantly different (P<0.05, ANOVA).</p

<i>Met</i> RNAi results in lethality during the larval-pupal-adult metamorphosis.

<p>dsRNA (10 µg per larva) was injected into selected larvae during initiation of the early wandering stage. Lethality was scored at the larval, prepupal, and pupal stages to compare the effects of <i>egfp</i>, <i>Met1</i>, <i>Met2</i>, and <i>Met1</i>+<i>2</i> dsRNAs.</p