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TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

Ghosh A, Rideout EJ, Grewal SS - PLoS Genet. (2014)

Bottom Line: When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth.This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle.Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, Health Research Innovation Center, Calgary, Alberta, Canada.

ABSTRACT
The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

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Muscle-specific TIF-IA inhibition reduces systemic insulin signaling.(A–C) Representative fat body images indicating FOXO (red) subcellular localization in (A) dMef2>+ (Fed), (B) dMef2>+ (Starved) and (C) dMef2>TIF-IA IR larvae, scale bar-500 µm. (D) Quantification indicating mean (N∶C, Nuclear∶Cytoplasmic) ratio of pixel intensity per fat body cell of dMef2>+ (Starved) (Grey bar, **P<0.001, One-way ANOVA and Tukey's post test) and dMef2>TIF-IA IR (White bar, *P<0.001, One-way ANOVA and Tukey's post test) animals, compared to fed control (dMef2>+) animals. 21 cells/genotype were scored. (E) qPCR indicates 4EBP mRNA levels were increased in dMef2>TIF-IA IR larvae compared to dMef2>+ control (*P = 0.002, Student's t-test). Data normalized to β tubulin mRNA. (F) Immunoblots indicate phospho Akt (Ser505), Akt and βtubulin levels in control (dMef2>+) and TIF-IA IR (dMef2>TIF-IA IR) larvae. (G) dMef2>TIF-IR larvae had reduced dILP3 mRNA (*P = 0.0003, Student's t-test) and dILP5 mRNA (*P = 0.015, Student's t-test) levels but dILP2 mRNA (P = 0.14, Student's t-test) levels were unaltered, compared to dMef2>+ control. Data normalized to β tubulin mRNA. (H–I) Representative images of larval brain insulin producing cells (IPC) at 96 hr AEL, indicating dILP2 protein accumulation of (H) dMef2>+ and (I) dMef2>TIF-IA IR animals, scale bar-20 µm. (J) Quantification showing mean pixel intensity/IPC cluster of dMef2>+ (n = 16) and dMef2>TIF-IA IR (n = 16) animals, n – number of IPC cluster assessed per genotype, images quantified with Image J software, (*P = 1.21×10−10, Student's t-test). (K) qPCR indicates Imp-L2 mRNA levels were induced in dMef2>TIF-IA IR larval muscle compared to dMef2>+ (control), (*P = 0.025, Student's t-test). Data normalized to β tubulin mRNA. All error bars indicate SEM.
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pgen-1004750-g005: Muscle-specific TIF-IA inhibition reduces systemic insulin signaling.(A–C) Representative fat body images indicating FOXO (red) subcellular localization in (A) dMef2>+ (Fed), (B) dMef2>+ (Starved) and (C) dMef2>TIF-IA IR larvae, scale bar-500 µm. (D) Quantification indicating mean (N∶C, Nuclear∶Cytoplasmic) ratio of pixel intensity per fat body cell of dMef2>+ (Starved) (Grey bar, **P<0.001, One-way ANOVA and Tukey's post test) and dMef2>TIF-IA IR (White bar, *P<0.001, One-way ANOVA and Tukey's post test) animals, compared to fed control (dMef2>+) animals. 21 cells/genotype were scored. (E) qPCR indicates 4EBP mRNA levels were increased in dMef2>TIF-IA IR larvae compared to dMef2>+ control (*P = 0.002, Student's t-test). Data normalized to β tubulin mRNA. (F) Immunoblots indicate phospho Akt (Ser505), Akt and βtubulin levels in control (dMef2>+) and TIF-IA IR (dMef2>TIF-IA IR) larvae. (G) dMef2>TIF-IR larvae had reduced dILP3 mRNA (*P = 0.0003, Student's t-test) and dILP5 mRNA (*P = 0.015, Student's t-test) levels but dILP2 mRNA (P = 0.14, Student's t-test) levels were unaltered, compared to dMef2>+ control. Data normalized to β tubulin mRNA. (H–I) Representative images of larval brain insulin producing cells (IPC) at 96 hr AEL, indicating dILP2 protein accumulation of (H) dMef2>+ and (I) dMef2>TIF-IA IR animals, scale bar-20 µm. (J) Quantification showing mean pixel intensity/IPC cluster of dMef2>+ (n = 16) and dMef2>TIF-IA IR (n = 16) animals, n – number of IPC cluster assessed per genotype, images quantified with Image J software, (*P = 1.21×10−10, Student's t-test). (K) qPCR indicates Imp-L2 mRNA levels were induced in dMef2>TIF-IA IR larval muscle compared to dMef2>+ (control), (*P = 0.025, Student's t-test). Data normalized to β tubulin mRNA. All error bars indicate SEM.

Mentions: The insulin pathway is the major endocrine regulator of body growth in larvae. Under nutrient-rich conditions, several dILPs are expressed and released into the larval hemolymph [33]. These dILPs then bind to a single insulin receptor in target cells and promote growth [26]. In contrast, starvation leads to reduced systemic insulin signaling and decreased growth. We therefore explored whether the growth inhibitory effects of muscle-specific TIF-IA knockdown were due to reduced systemic insulin signaling. Under nutrient rich conditions, high level of insulin signaling leads to activation of Akt kinase and phosphorylation and nuclear exclusion of the FOXO transcription factor. But when insulin signaling is reduced, FOXO relocalizes to the nucleus and activates target genes such as eIF4E-Binding Protein (4EBP). Therefore, changes in FOXO nuclear localization and transcriptional activity serve as a reliable ‘read-out’ of insulin signaling [34]–[36]. As previously reported, we found that FOXO was excluded from nuclei in fat body cells from fed larvae (Figure 5A), but showed strong nuclear accumulation in fat body cells from starved larvae (Figure 5B). When we knocked-down TIF-IA levels in muscle (dMef2>TIF-IA IR), FOXO showed strong, statistically significant nuclear accumulation in fat body cells (Figure 5C, D). We next measured the levels of 4EBP, a FOXO target gene, and found that dMef2>TIF-IA IR larvae had increased 4EBP mRNA levels with respect to control (dMef2>+) larvae (Figure 5E). Finally we measured the examined levels of phosphorylated Akt – the kinase downstream of insulin signaling that is responsible for phosphorylation and inhibition of FOXO. Using western blotting with an anti-phospho Akt (Ser505) antibody, we found that dMef2>TIF-IA IR had markedly reduced levels of phospho Akt compared to control (dMef2>+) larvae (Figure 5F). Levels of total Akt were also lower, but much less so than the suppression in levels of phosphorylated Akt. Together these data suggest that TIF-IA knockdown in muscle leads to reduction in systemic insulin signaling.


TIF-IA-dependent regulation of ribosome synthesis in drosophila muscle is required to maintain systemic insulin signaling and larval growth.

Ghosh A, Rideout EJ, Grewal SS - PLoS Genet. (2014)

Muscle-specific TIF-IA inhibition reduces systemic insulin signaling.(A–C) Representative fat body images indicating FOXO (red) subcellular localization in (A) dMef2>+ (Fed), (B) dMef2>+ (Starved) and (C) dMef2>TIF-IA IR larvae, scale bar-500 µm. (D) Quantification indicating mean (N∶C, Nuclear∶Cytoplasmic) ratio of pixel intensity per fat body cell of dMef2>+ (Starved) (Grey bar, **P<0.001, One-way ANOVA and Tukey's post test) and dMef2>TIF-IA IR (White bar, *P<0.001, One-way ANOVA and Tukey's post test) animals, compared to fed control (dMef2>+) animals. 21 cells/genotype were scored. (E) qPCR indicates 4EBP mRNA levels were increased in dMef2>TIF-IA IR larvae compared to dMef2>+ control (*P = 0.002, Student's t-test). Data normalized to β tubulin mRNA. (F) Immunoblots indicate phospho Akt (Ser505), Akt and βtubulin levels in control (dMef2>+) and TIF-IA IR (dMef2>TIF-IA IR) larvae. (G) dMef2>TIF-IR larvae had reduced dILP3 mRNA (*P = 0.0003, Student's t-test) and dILP5 mRNA (*P = 0.015, Student's t-test) levels but dILP2 mRNA (P = 0.14, Student's t-test) levels were unaltered, compared to dMef2>+ control. Data normalized to β tubulin mRNA. (H–I) Representative images of larval brain insulin producing cells (IPC) at 96 hr AEL, indicating dILP2 protein accumulation of (H) dMef2>+ and (I) dMef2>TIF-IA IR animals, scale bar-20 µm. (J) Quantification showing mean pixel intensity/IPC cluster of dMef2>+ (n = 16) and dMef2>TIF-IA IR (n = 16) animals, n – number of IPC cluster assessed per genotype, images quantified with Image J software, (*P = 1.21×10−10, Student's t-test). (K) qPCR indicates Imp-L2 mRNA levels were induced in dMef2>TIF-IA IR larval muscle compared to dMef2>+ (control), (*P = 0.025, Student's t-test). Data normalized to β tubulin mRNA. All error bars indicate SEM.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214618&req=5

pgen-1004750-g005: Muscle-specific TIF-IA inhibition reduces systemic insulin signaling.(A–C) Representative fat body images indicating FOXO (red) subcellular localization in (A) dMef2>+ (Fed), (B) dMef2>+ (Starved) and (C) dMef2>TIF-IA IR larvae, scale bar-500 µm. (D) Quantification indicating mean (N∶C, Nuclear∶Cytoplasmic) ratio of pixel intensity per fat body cell of dMef2>+ (Starved) (Grey bar, **P<0.001, One-way ANOVA and Tukey's post test) and dMef2>TIF-IA IR (White bar, *P<0.001, One-way ANOVA and Tukey's post test) animals, compared to fed control (dMef2>+) animals. 21 cells/genotype were scored. (E) qPCR indicates 4EBP mRNA levels were increased in dMef2>TIF-IA IR larvae compared to dMef2>+ control (*P = 0.002, Student's t-test). Data normalized to β tubulin mRNA. (F) Immunoblots indicate phospho Akt (Ser505), Akt and βtubulin levels in control (dMef2>+) and TIF-IA IR (dMef2>TIF-IA IR) larvae. (G) dMef2>TIF-IR larvae had reduced dILP3 mRNA (*P = 0.0003, Student's t-test) and dILP5 mRNA (*P = 0.015, Student's t-test) levels but dILP2 mRNA (P = 0.14, Student's t-test) levels were unaltered, compared to dMef2>+ control. Data normalized to β tubulin mRNA. (H–I) Representative images of larval brain insulin producing cells (IPC) at 96 hr AEL, indicating dILP2 protein accumulation of (H) dMef2>+ and (I) dMef2>TIF-IA IR animals, scale bar-20 µm. (J) Quantification showing mean pixel intensity/IPC cluster of dMef2>+ (n = 16) and dMef2>TIF-IA IR (n = 16) animals, n – number of IPC cluster assessed per genotype, images quantified with Image J software, (*P = 1.21×10−10, Student's t-test). (K) qPCR indicates Imp-L2 mRNA levels were induced in dMef2>TIF-IA IR larval muscle compared to dMef2>+ (control), (*P = 0.025, Student's t-test). Data normalized to β tubulin mRNA. All error bars indicate SEM.
Mentions: The insulin pathway is the major endocrine regulator of body growth in larvae. Under nutrient-rich conditions, several dILPs are expressed and released into the larval hemolymph [33]. These dILPs then bind to a single insulin receptor in target cells and promote growth [26]. In contrast, starvation leads to reduced systemic insulin signaling and decreased growth. We therefore explored whether the growth inhibitory effects of muscle-specific TIF-IA knockdown were due to reduced systemic insulin signaling. Under nutrient rich conditions, high level of insulin signaling leads to activation of Akt kinase and phosphorylation and nuclear exclusion of the FOXO transcription factor. But when insulin signaling is reduced, FOXO relocalizes to the nucleus and activates target genes such as eIF4E-Binding Protein (4EBP). Therefore, changes in FOXO nuclear localization and transcriptional activity serve as a reliable ‘read-out’ of insulin signaling [34]–[36]. As previously reported, we found that FOXO was excluded from nuclei in fat body cells from fed larvae (Figure 5A), but showed strong nuclear accumulation in fat body cells from starved larvae (Figure 5B). When we knocked-down TIF-IA levels in muscle (dMef2>TIF-IA IR), FOXO showed strong, statistically significant nuclear accumulation in fat body cells (Figure 5C, D). We next measured the levels of 4EBP, a FOXO target gene, and found that dMef2>TIF-IA IR larvae had increased 4EBP mRNA levels with respect to control (dMef2>+) larvae (Figure 5E). Finally we measured the examined levels of phosphorylated Akt – the kinase downstream of insulin signaling that is responsible for phosphorylation and inhibition of FOXO. Using western blotting with an anti-phospho Akt (Ser505) antibody, we found that dMef2>TIF-IA IR had markedly reduced levels of phospho Akt compared to control (dMef2>+) larvae (Figure 5F). Levels of total Akt were also lower, but much less so than the suppression in levels of phosphorylated Akt. Together these data suggest that TIF-IA knockdown in muscle leads to reduction in systemic insulin signaling.

Bottom Line: When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth.This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle.Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, and Clark H. Smith Brain Tumour Centre, Southern Alberta Cancer Research Institute, University of Calgary, Health Research Innovation Center, Calgary, Alberta, Canada.

ABSTRACT
The conserved TOR kinase signaling network links nutrient availability to cell, tissue and body growth in animals. One important growth-regulatory target of TOR signaling is ribosome biogenesis. Studies in yeast and mammalian cell culture have described how TOR controls rRNA synthesis-a limiting step in ribosome biogenesis-via the RNA Polymerase I transcription factor TIF-IA. However, the contribution of TOR-dependent ribosome synthesis to tissue and body growth in animals is less clear. Here we show in Drosophila larvae that ribosome synthesis in muscle is required non-autonomously to maintain normal body growth and development. We find that amino acid starvation and TOR inhibition lead to reduced levels of TIF-IA, and decreased rRNA synthesis in larval muscle. When we mimic this decrease in muscle ribosome synthesis using RNAi-mediated knockdown of TIF-IA, we observe delayed larval development and reduced body growth. This reduction in growth is caused by lowered systemic insulin signaling via two endocrine responses: reduced expression of Drosophila insulin-like peptides (dILPs) from the brain and increased expression of Imp-L2-a secreted factor that binds and inhibits dILP activity-from muscle. We also observed that maintaining TIF-IA levels in muscle could partially reverse the starvation-mediated suppression of systemic insulin signaling. Finally, we show that activation of TOR specifically in muscle can increase overall body size and this effect requires TIF-IA function. These data suggest that muscle ribosome synthesis functions as a nutrient-dependent checkpoint for overall body growth: in nutrient rich conditions, TOR is required to maintain levels of TIF-IA and ribosome synthesis to promote high levels of systemic insulin, but under conditions of starvation stress, reduced muscle ribosome synthesis triggers an endocrine response that limits systemic insulin signaling to restrict growth and maintain homeostasis.

Show MeSH
Related in: MedlinePlus