Research ArticleCELL BIOLOGY

Octopamine connects nutrient cues to lipid metabolism upon nutrient deprivation

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Science Advances  06 May 2016:
Vol. 2, no. 5, e1501372
DOI: 10.1126/sciadv.1501372
  • Fig. 1 Biosynthesis of octopamine is up-regulated during starvation.

    (A) Expression of Ptbh-1::gfp was up-regulated in starved worms relative to well-fed worms (in the presence of E. coli OP50). The right panel shows quantification of GFP levels. (B) The mRNA levels of tbh-1 were determined by qRT-PCR. (C) The levels of octopamine in starved worms were higher than those in well-fed worms. Results are means ± SD of three experiments. *P < 0.05 versus well-fed worms. (D) Survival rate of tbh-1(n3247) worms was lower than that of wild-type (WT) worms after 3 days of starvation. Exogenous octopamine (OA; 1 mM) markedly restored the survival of tbh-1(n3247) worms. Results are means ± SD of three experiments. aP < 0.05, tbh-1(n3247) versus WT worms; bP < 0.05, tbh-1 + octopamine versus tbh-1.

  • Fig. 2 Octopamine promotes lipid hydrolysis by up-regulating the expression of the lipase gene lips-6.

    (A and B) Lipid mobilization was regulated by octopamine. (A) The quantity of lipid droplets determined by Oil Red O staining in WT worms was significantly lower than that in tbh-1(n3247) worms after 24 hours of starvation. Octopamine (1 mM) treatment promoted lipid hydrolysis in either starved tbh-1(n3247) worms or well-fed WT worms. The right panel represents relative Oil Red O intensity. (B) The relative TAG contents were determined by GC-MS. Results are means ± SD of three experiments. TL, total lipid. (C and D) The mRNA levels of lips-6 (C) and the expression of Plips-6::gfp (D) were up-regulated by octopamine after 12 hours of starvation. Results are means ± SD of three experiments. aP < 0.05 versus well-fed WT worms; bP < 0.05 versus starved WT worms; cP < 0.05 versus well-fed tbh-1(n3247) worms; dP < 0.05 versus starved tbh-1(n3247) worms. (E) Lipid hydrolysis was suppressed by lips-6 RNAi after 24 hours of starvation. However, exogenous octopamine (1 mM) failed to restore lipid hydrolysis in lips-6 (RNAi) worms. The right panel represents relative Oil Red O intensity. Results are means ± SD of three experiments. aP < 0.05 versus well-fed worms + empty vector (EV); bP < 0.05 versus starved worms + empty vector.

  • Fig. 3 Octopamine promotes lipid hydrolysis via its receptor SER-3.

    (A and B) Lipid hydrolysis was inhibited in ser-3(ad1774), but not ser-6(tm2146) or octr-1(ok371), mutants after 24 hours of starvation. (A) Oil Red O staining. The right panel represents relative Oil Red O intensity. (B) The relative TAG contents were determined by GC-MS. Results are means ± SD of three experiments. (C) The mRNA levels of lips-6 were also reduced in ser-3(ad1774), but not ser-6(tm2146) or octr-1(ok371), worms during starvation. Results are means ± SD of three experiments. aP < 0.05 versus well-fed WT worms; bP < 0.05 versus starved WT worms. (D and E) Intestinal-specific knockdown of ser-3 by RNAi reduced lipid hydrolysis (D) and the expression of lips-6 (E). Results are means ± SD of three experiments. aP < 0.05 versus well-fed worms + empty vector; bP < 0.05 versus starved worms + empty vector. (F and G) Expression of ser-3 under the control of the intestinal ges-1 markedly restored lipid hydrolysis (F) and the expression of lips-6 (G) in ser-3(ad1774) mutants. Results are means ± SD of three experiments. *P < 0.05 versus empty vector.

  • Fig. 4 DAF-12/DIN-1 up-regulates the expression of tbh-1 during starvation.

    (A) tbh-1 promoters with three AGTACA hexamer elements (−4139, −3193, and −2711 upstream of ATG) for DAF-12. Mutagenesis or deletion of these elements led to a significant reduction in Ptbh-1::gfp expression during food-deprived conditions. (B to D) A mutation in daf-12(rh61rh411) or din-1(dh127) inhibited the expression of Ptbh-1::gfp expression (B) and reduced the mRNA levels of tbh-1 (C) and the levels of octopamine (D) in starved worms. The right part shows quantification of GFP levels (B). Results are means ± SD of three experiments. aP < 0.01 versus well-fed WT worms; bP < 0.05 versus starved WT worms. (E) The three putative DAF-12 binding sites in the promoter regions of tbh-1 were detected by ChIP with anti-GFP antibody. ChIP results were quantitated by qPCR. *P < 0.05 versus well-fed worms.

  • Fig. 5 DAF-12/DIN-1 confers resistance to starvation.

    (A) Mutation in daf-12(rh61rh411) or din-1(dh127) suppressed lipid hydrolysis determined by Oil Red O staining after 24 hours of starvation. Application of octopamine (1 mM) restored fasting-induced breakdown. The right panel represents relative Oil Red O intensity. Results are means ± SD of three experiments. (B and C) Expression of lips-6 in WT, daf-12(rh61rh411), and din-1(dh127) worms after 12 hours of starvation. (B) lips-6 mRNA levels. (C) Expression of Plips-6::gfp. The right part shows quantification of GFP levels. Results are means ± SD of three experiments. aP < 0.05 versus well-fed WT worms; bP < 0.05 versus starved WT worms; cP < 0.05 versus well-fed daf-12(rh61rh411) or din-1(dh127) worms; dP < 0.05 versus starved daf-12(rh61rh411) or din-1(dh127) worms. (D and E) Expression of daf-12 under the control of the tbh-1 promoter restored lipid hydrolysis (D) and the expression of lips-6 (E) in daf-12(rh61rh411) animals. The lower panel represents relative Oil Red O intensity (D). Results are means ± SD of three experiments. *P < 0.05 versus empty vector.

  • Fig. 6 A proposed model for octopamine-mediated starvation resistance.

    Under nutrient deprivation, the liganded DAF-12 shifts the equilibrium to the unliganded DAF-12/DIN-1 complex. The latter promotes the octopamine biosynthesis by up-regulating the expression of tbh-1 in the RIC neurons. Octopamine, in turn, activates its receptor SER-3 to up-regulate the expression of the lipase gene lips-6 in the intestine. Concomitantly, LIPS-6 contributes to lipid hydrolysis to maintain energy balance.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/5/e1501372/DC1

    fig. S1. LC-MS/MS detection of octopamine contents in worms.

    fig. S2. TLC analysis of total lipids extracted from worms.

    fig. S3. Overexpression of tbh-1 enhances lipid hydrolysis in well-fed wild-type worms.

    fig. S4. Starvation does not alter the expression of atgl-1.

    fig. S5. A mutation in tbh-1(n3247) does not affect the expression of fil-1 or fil-2 in starved worms.

    fig. S6. Knockdown of lips-6 leads to reduced lips-6 expression.

    fig. S7. Overexpression of lips-6 in the intestine promotes lipid hydrolysis in well-fed worms.

    fig. S8. Octopamine fails to restore lipid hydrolysis and expression of lips-6 in ser-3(ad1774) mutants during starvation.

    fig. S9. SER-3 in the neurons and muscle is not involved in lipid hydrolysis during starvation.

    fig. S10. SER-3 is involved in resistance to starvation.

    table S1. ChIP-qPCR analysis of the tbh-1 promoter.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. LC-MS/MS detection of octopamine contents in worms.
    • fig. S2. TLC analysis of total lipids extracted from worms.
    • fig. S3. Overexpression of tbh-1 enhances lipid hydrolysis in well-fed wild-type worms.
    • fig. S4. Starvation does not alter the expression of atgl-1.
    • fig. S5. A mutation in tbh-1(n3247) does not affect the expression of fil-1 or fil-2 in starved worms.
    • fig. S6. Knockdown of lips-6 by RNAi leads to reduced lips-6 expression.
    • fig. S7. Overexpression of lips-6 in the intestine promotes lipid hydrolysis in well-fed WT worms.
    • fig. S8. Octopamine fails to restore lipid hydrolysis and expression of lips-6 in ser-3(ad1774) mutants during starvation.
    • fig. S9. SER-3 in the neurons and muscle is not involved in lipid hydrolysis during starvation.
    • fig. S10. SER-3 is involved in resistance to starvation.
    • table S1. ChIP-qPCR analysis of the tbh-1 promoter.

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