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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 Expressed at a Higher Level in AMPK-deficient Animals during the Early Dauer Stage.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to restrictive temperature (25°C) at the L1 stage (see materials and methods). Scale bar = 10μm. (B) Western blot analysis of GFP levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (C) Western blot analysis of endogenous ATGL-1 levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (D) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 4 animals using semi-quantitative RT-PCR. act-1 was used as loading control.
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pone.0130480.g004: ATGL-1::GFP Expressed at a Higher Level in AMPK-deficient Animals during the Early Dauer Stage.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to restrictive temperature (25°C) at the L1 stage (see materials and methods). Scale bar = 10μm. (B) Western blot analysis of GFP levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (C) Western blot analysis of endogenous ATGL-1 levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (D) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 4 animals using semi-quantitative RT-PCR. act-1 was used as loading control.

Mentions: We also documented the ATGL-1::GFP levels (Fig 4A and 4B) and the endogenous ATGL-1 levels (Fig 4C) in the same animals during the early dauer stage from dauer day 1 (72 hours after shifting to restrictive temperature since L1 stage) to 4 and found that, similar to the dauer entry period, both levels were always more abundant in the larvae that lacked functional AMPK. Furthermore, the mRNA level of ATGL-1::GFP was also unchanged in control daf-2 and daf-2; aak(0) dauer day 4 animals (Fig 4D). Therefore ATGL-1 levels are unlikely to be subject to feedback regulation and most probably increase in the absence of AMPK through post-transcriptional mechanisms. Taken together these data suggest that ATGL-1 is phosphorylated by AMPK, and this post-translational modification affects the enzyme by decreasing its stability.


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 Expressed at a Higher Level in AMPK-deficient Animals during the Early Dauer Stage.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to restrictive temperature (25°C) at the L1 stage (see materials and methods). Scale bar = 10μm. (B) Western blot analysis of GFP levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (C) Western blot analysis of endogenous ATGL-1 levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (D) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 4 animals using semi-quantitative RT-PCR. act-1 was used as loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476745&req=5

pone.0130480.g004: ATGL-1::GFP Expressed at a Higher Level in AMPK-deficient Animals during the Early Dauer Stage.(A) Comparison of ATGL-1::GFP levels between control daf-2 and daf-2; aak(0) animals during the early dauer stage. Dauer day 1 is defined as 72 hours after shifting to restrictive temperature (25°C) at the L1 stage (see materials and methods). Scale bar = 10μm. (B) Western blot analysis of GFP levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (C) Western blot analysis of endogenous ATGL-1 levels in control daf-2 and daf-2; aak(0) mutant animals during early dauer stage. (D) Quantification of ATGL-1::GFP mRNA levels in control daf-2 and daf-2; aak(0) mutant dauer day 4 animals using semi-quantitative RT-PCR. act-1 was used as loading control.
Mentions: We also documented the ATGL-1::GFP levels (Fig 4A and 4B) and the endogenous ATGL-1 levels (Fig 4C) in the same animals during the early dauer stage from dauer day 1 (72 hours after shifting to restrictive temperature since L1 stage) to 4 and found that, similar to the dauer entry period, both levels were always more abundant in the larvae that lacked functional AMPK. Furthermore, the mRNA level of ATGL-1::GFP was also unchanged in control daf-2 and daf-2; aak(0) dauer day 4 animals (Fig 4D). Therefore ATGL-1 levels are unlikely to be subject to feedback regulation and most probably increase in the absence of AMPK through post-transcriptional mechanisms. Taken together these data suggest that ATGL-1 is phosphorylated by AMPK, and this post-translational modification affects the enzyme by decreasing its stability.

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.