GBP1 and GBP2 regulate ILP2 and ILP5 secretion.

<p><b>(A, B)</b> Knocking down GBP1 and GBP2 increases ILP2 (A) and ILP5 (B) accumulation in the insulin-producing cells. The scale bars are 20 μm. <b>(C, D)</b> Knocking down GBP1 and GBP2 increases the densities of ILP2 (C) and ILP5 (D) signals in the insulin-producing cells. We standardized the densities of ILPs by fixing the values from <i>C7</i>>2xGFP to 1. <i>n</i> = 16–27 for both ILP2 and ILP5. <b>(E, F)</b> The <i>gbp1</i>, <i>gbp2 ex67</i> mutant shows increased ILP2 (E) and ILP5 (F) accumulation in the insulin-producing cells. The scale bars are 20 μm. <b>(G, H)</b> The <i>gbp1</i>, <i>gbp2 ex67</i> mutant shows increased densities of ILP2 (G) and ILP5 (H) signals in the insulin-producing cells. We standardized the densities of ILPs by fixing the values from w1118 larvae to 1. <i>n</i> = 34–41 for both ILP2 and ILP5. Treatments sharing the same letter indicate the groups that are statistically indistinguishable from one another (ANOVA and pairwise <i>t</i> tests, <i>p</i> < 0.05). The supplementary file in which the data used to generate each plot can be found is <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002392#pbio.1002392.s001" target="_blank">S1 Data</a>.</p

GBP1 and GBP2 are downstream of TOR signaling.

<p><b>(A, B)</b> Overexpressing either GBP1 or GBP2 partially rescues body size reduction in females (A) and males (B) with reduced TOR signaling in their fat bodies. <i>n</i> = 47–64 for A and 59–67 for B. <b>(C)</b> Overexpressing either GBP1 or GBP2 partially increases growth rate in larvae with reduced TOR signaling in the fat body. <i>n</i> = 14-18/time point. <b>(D)</b> Overexpressing either GBP1 or GBP2 partially rescues the duration of the L3 in TOR signaling reduced larvae. <i>n</i> = 108–129. <b>(E, F)</b> Overexpressing either GBP1 or GBP2 reduces ILP2 (E) and ILP5 (F) accumulation in the insulin-producing cells. <b>(G, H)</b> Overexpressing GBP1 or GBP2 reduces the densities of ILP2 (G) and ILP5 (H) signals in the insulin-producing cells. We standardized the densities of ILPs by fixing the values from <i>C7</i>>GFP, TSC1, TSC2 to 1. <i>n</i> = 30–42 for both ILP2 and ILP5. <b>(I)</b> Overexpressing either GBP1 or GBP2 reduces FRE-luciferase activity in the entire body. <i>n</i> = 5. One copy of UAS transgene, UAS TSC1 and UAS TSC2 (three UAS in total) were co-expressed using the <i>C7</i> Gal4 driver for all experiments. For the wild-type control, we overexpressed three copies of UAS GFP using the <i>C7</i> Gal4 driver. Treatments sharing the same letter indicate the groups that are statistically indistinguishable from one another (ANOVA and pairwise <i>t</i> tests, <i>p</i> < 0.05). Growth rate was analyzed by ANCOVA and post hoc comparisons of the slopes. The supplementary file in which the data used to generate each plot can be found is <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002392#pbio.1002392.s001" target="_blank">S1 Data</a>.</p

Both GBP1 and GBP2 are secreted from the fat body and directly act on the brain to induce ILP2 and ILP5 secretion.

<p><b>(A, B)</b> Culturing wild-type brains with conditioned media from GBP1 and GBP2 expressing cells induces low level of ILP2 (A) and ILP5 (B) accumulation in the insulin-producing cells. To obtain brains, w1118 larvae staged at the onset of the L3 were fed on normal food for 12 h followed by 12 h incubation on 1% non-nutritive agar to induce strong ILP accumulation. To make conditioned media, an equal amount of Actin Gal4 plasmid and either UAS <i>gbp1</i>, UAS <i>gbp2</i>, or UAS <i>egfp</i> plasmid were transfected into the <i>Drosophila</i> SL2/DL2 cell line. Forty-eight hours after transfection, the media were centrifuged and the supernatant was diluted with culture medium to 100%, 50% and 25% the original concentrations. The scale bars are 20 μm. <b>(C, D)</b> Culturing wild-type brains with conditioned media from GBP1 and GBP2 expressing cells decreases the densities of ILP2 (C) and ILP5 (D) signals in the insulin-producing cells. We standardized the densities of ILPs by fixing the values from GFP 100% to 1. <i>n</i> = 16–27 for both ILP2 and ILP5. <b>(E, F)</b> Co-culturing wild-type brains with fat bodies from wild-type, but not from <i>gbp1</i>, <i>gbp2 ex67</i>, larvae induces low levels of ILP2 (E) and ILP5 (F) accumulation in the insulin-producing cells. The brains were obtained as above. The fat bodies were obtained as described in the Materials and Methods. <b>(G, H)</b> Co-culturing wild-type brains with fat bodies from wild-type, but not from <i>gbp1</i>, <i>gbp2 ex67</i>, larvae decreases the densities of ILP2 (G) and ILP5 (H) signals in the insulin-producing cells. We standardized the densities of ILPs by fixing the values from the “no fat body” treatment (culturing with a plain medium) to 1. <i>n</i> = 12–22 for both ILP2 and ILP5. Treatments sharing the same letter indicate the groups that are statistically indistinguishable from one another (ANOVA and pairwise <i>t</i> tests, <i>p</i> < 0.05). The supplementary file in which the data used to generate each plot can be found is <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002392#pbio.1002392.s001" target="_blank">S1 Data</a>.</p

Overexpressing GBP1 and GBP2 in the fat body rescues body size in <i>gbp1</i>, <i>gbp2 ex67</i> null mutant larvae.

<p><b>(A, B)</b> Overexpressing both GBP1 and GBP2 partially rescues body size reduction in <i>gbp1</i>, <i>gbp2 ex67</i> mutant females (A) and males (B) at 22°C. <i>n</i> = 51–52 for A and <i>n</i> = 54–65 for B. <b>(C)</b> Overexpressing both GBP1 and GBP2 partially increases growth rate in <i>gbp1</i>, <i>gbp2 ex67</i> mutant larvae. <i>n</i> = 14–20/time point. <b>(D)</b> Overexpressing both GBP1 and GBP2 partially rescues the duration of the L3 in <i>gbp1</i>, <i>gbp2 ex67</i> mutant larvae. <i>n</i> = 111–119. <b>(E, F)</b> Overexpressing both GBP1 and GBP2 reduces ILP2 (E) and ILP5 (F) accumulation in the insulin-producing cells of <i>gbp1</i>, <i>gbp2 ex67</i> mutant larvae. Larvae were staged at the onset of the L3, and then fed on normal food for 24 h. The insulin-producing cells were immunostained using an anti-ILP2 antibody and an anti-ILP5 antibody. <b>(G, H)</b> Overexpressing both GBP1 and GBP2 reduces the densities of ILP2 (G) and ILP5 (H) signals in the insulin-producing cells. The densities of ILP2 and ILP5 were quantified using ImageJ. We standardized the densities of ILPs by fixing the values from <i>C7</i>>GFP in the <i>gbp1</i>, <i>gbp2 ex67</i> mutant background to 1. <i>n</i> = 30–60. <b>(I)</b> Overexpressing both GBP1 and GBP2 reduces FRE-luciferase activity in the entire body of <i>gbp1</i>, <i>gbp2 ex67</i> mutant larvae. <i>n</i> = 5. Two copies of UAS transgenes were expressed using the <i>C7</i> Gal4 driver. For the wild-type control, we overexpressed two copies of UAS GFP using the <i>C7</i> Gal4 driver. Treatments sharing the same letter indicate the groups that are statistically indistinguishable from one another (ANOVA and pairwise <i>t</i> tests, <i>p</i> < 0.05). Growth rate was analyzed by ANCOVA and post hoc comparisons of the slopes. The supplementary file in which the data used to generate each plot can be found is <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002392#pbio.1002392.s001" target="_blank">S1 Data</a>.</p

Nutrition regulates <i>gbp1</i> and <i>gbp2</i> expression via TOR signaling in the fat body.

<p><b>(A, B)</b> Amino acid intake is sufficient to induce <i>gbp1</i> (A) and <i>gbp2</i> (B) mRNA expression in the fat body of w1118 larvae. Larvae were staged at the onset of the L3, and fed on normal food for 12 h. Then they were starved for 12 h on 1% non-nutritive agar followed by an additional 12 h on one of five nutritionally different media. Columns sharing the same letter indicate the groups that are statistically indistinguishable from one another (ANOVA and pairwise <i>t</i> tests, <i>p</i> < 0.05). <b>(C, D)</b> Reducing TOR signaling activity in the fat body decreases <i>gbp1</i> (C) and <i>gbp2</i> (D) mRNA expression in the fat body. Larvae were staged at the onset of the L3, and fed on normal food for 24 h. Numbers indicate p-values (ANOVA and pairwise <i>t</i> tests). We normalized the values using an internal control, <i>RpL3</i>. Then, we standardized the expression level of each gene by fixing the values from non-nutritive agar treated animals to 1 in A and B, and from <i>C7</i>>2xGFP to 1 in C and D. We used the fat bodies from five larvae for each sample and five biologically independent samples for each condition. Each bar indicates the relative mean expression ± SEM. The supplementary file in which the data used to generate each plot can be found is <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002392#pbio.1002392.s001" target="_blank">S1 Data</a>.</p

Changing TOR signaling activity in the fat body affects body size.

<p><b>(A, B)</b> Changing TOR signaling in the fat body affects final body size in females (A) and males (B). <i>n</i> = 47–72 for A and <i>n</i> = 44–70 for B. Pharate adults (approximately 2–14 h before eclosion) were weighed as a proxy of final adult size. <b>(C)</b> Activating TOR signaling in the fat body does not affect growth rate while repressing TOR signaling reduces growth rate. <i>n</i> = 14–17/time point. <b>(D)</b> Activating TOR signaling in the fat body slightly shortens the duration of the L3. Repressing TOR signaling in the fat body extends the duration of the L3. <i>n</i> = 77–113. Two copies of UAS transgenes were expressed using the <i>C7</i> Gal4 driver in all experiments. Treatments sharing the same letter indicate the groups that are statistically indistinguishable from one another (<i>p</i> < 0.05, ANOVA and pairwise <i>t</i> tests). Growth rate was analyzed by ANCOVA and post hoc comparisons of the slopes. The supplementary file in which the data used to generate each plot can be found is <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002392#pbio.1002392.s001" target="_blank">S1 Data</a>.</p

Final weight all

Pharate adult weights for males and females in all genotypes examined

Ecdysone quantification final

Ecdysone quantifications for P0206>, >FoxO, P0206>FoxO and P0206>FoxO NK. All genotypes are in a FoxO null background

Real-Time PCR final

Real-Time PCR quantification of E74B, phm and di

Proposed mechanism of Vvl action.

<p>Vvl influences JH production via <i>jhamt</i> expression and hence the timing of metamorphosis. Vvl also regulates the biosynthesis of ecdysteroids and the molting pathway.</p