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AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae.

Xie M, Roy R - PLoS ONE (2015)

Bottom Line: Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation.In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation.Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 1205 avenue Docteur Penfield, Montreal, Canada.

ABSTRACT
Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

No MeSH data available.


ATGL-1::GFP Accumulates to Higher Levels in AMPK-deficient Animals Prior to Dauer Entry.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the dauer entry period. Images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x20 magnification. All strains harbor the same hjIs67[Patgl-1::atgl-1::GFP] transgenic array in (A) and (B). Scale bar = 20μm. (B) Western blot analysis of ATGL-1::GFP levels as measured using an anti-GFP antibody in control daf-2 and daf-2; aak(0) mutant animals during the period prior to dauer entry. (C) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 0 animals using semi-quantitative RT-PCR. act-1 was used as loading control.
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pone.0130480.g002: ATGL-1::GFP Accumulates to Higher Levels in AMPK-deficient Animals Prior to Dauer Entry.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the dauer entry period. Images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x20 magnification. All strains harbor the same hjIs67[Patgl-1::atgl-1::GFP] transgenic array in (A) and (B). Scale bar = 20μm. (B) Western blot analysis of ATGL-1::GFP levels as measured using an anti-GFP antibody in control daf-2 and daf-2; aak(0) mutant animals during the period prior to dauer entry. (C) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 0 animals using semi-quantitative RT-PCR. act-1 was used as loading control.

Mentions: To discern whether AMPK regulates ATGL-1 during the dauer stage through a possible allosteric effect of the phosphorylation versus an effect on ATGL-1 stability or localization, we monitored the fate of ATGL-1 after being phosphorylated by AMPK during this stage. Because AMPK phosphorylation can often trigger proteasome-mediated degradation [19], we wondered whether this possibility could explain the AMPK-mediated reduction in ATGL-1 function during the dauer stage. We therefore introduced a fully functional ATGL-1::GFP translational fusion protein into control daf-2 and daf-2; aak(0) dauer larvae to compare ATGL-1 expression levels in these animals. We documented the ATGL-1::GFP expression in these animals at various time points during the entire dauer entry period, which we define here as the first 48 hours after being shifted to restrictive temperature (Fig 2A). ATGL-1::GFP was significantly more abundant in the absence of AMPK at all the time points during the entire dauer entry period (Fig 2B).


AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae.

Xie M, Roy R - PLoS ONE (2015)

ATGL-1::GFP Accumulates to Higher Levels in AMPK-deficient Animals Prior to Dauer Entry.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the dauer entry period. Images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x20 magnification. All strains harbor the same hjIs67[Patgl-1::atgl-1::GFP] transgenic array in (A) and (B). Scale bar = 20μm. (B) Western blot analysis of ATGL-1::GFP levels as measured using an anti-GFP antibody in control daf-2 and daf-2; aak(0) mutant animals during the period prior to dauer entry. (C) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 0 animals using semi-quantitative RT-PCR. act-1 was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4476745&req=5

pone.0130480.g002: ATGL-1::GFP Accumulates to Higher Levels in AMPK-deficient Animals Prior to Dauer Entry.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the dauer entry period. Images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x20 magnification. All strains harbor the same hjIs67[Patgl-1::atgl-1::GFP] transgenic array in (A) and (B). Scale bar = 20μm. (B) Western blot analysis of ATGL-1::GFP levels as measured using an anti-GFP antibody in control daf-2 and daf-2; aak(0) mutant animals during the period prior to dauer entry. (C) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 0 animals using semi-quantitative RT-PCR. act-1 was used as loading control.
Mentions: To discern whether AMPK regulates ATGL-1 during the dauer stage through a possible allosteric effect of the phosphorylation versus an effect on ATGL-1 stability or localization, we monitored the fate of ATGL-1 after being phosphorylated by AMPK during this stage. Because AMPK phosphorylation can often trigger proteasome-mediated degradation [19], we wondered whether this possibility could explain the AMPK-mediated reduction in ATGL-1 function during the dauer stage. We therefore introduced a fully functional ATGL-1::GFP translational fusion protein into control daf-2 and daf-2; aak(0) dauer larvae to compare ATGL-1 expression levels in these animals. We documented the ATGL-1::GFP expression in these animals at various time points during the entire dauer entry period, which we define here as the first 48 hours after being shifted to restrictive temperature (Fig 2A). ATGL-1::GFP was significantly more abundant in the absence of AMPK at all the time points during the entire dauer entry period (Fig 2B).

Bottom Line: Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation.In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation.Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, McGill University, 1205 avenue Docteur Penfield, Montreal, Canada.

ABSTRACT
Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

No MeSH data available.