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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.


AMPK Regulates ATGL-1 Association with Lipid Droplets in Dauer Larvae.(A)-(B) Signal overlap between ATGL-1::GFP (Green) and C1-BODIPY-C12-stained lipid droplets (Red) was compared in control daf-2 and daf-2; aak(0) mutant animals at 32 (A) and 48 hours (B) after shifting to restricted temperature. ATGL-1::GFP signal was closely associated with the labeled lipid droplets in daf-2; aak(0) mutant animals (white arrowheads in the insets) while the signals are clearly distinguishable from each other in control daf-2 animals. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified by the same magnification. (C) Western blot analysis of the endogenous ATGL-1 levels in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 (48 hours after shifting to restricted temperature) dauer extracts of control daf-2 and daf-2; aak(0) mutant animals. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level according to the recent proteomic study on C. elegans lipid droplets [39]. (D) Lipid droplet isolation method verified by significant C1-BODIPY-C12 staining and triglyceride enrichment in the isolated lipid droplets portion comparing to the cytoplasm (remaining portion of the total lysate) from daf-2 day 0 dauer larvae.
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pone.0130480.g006: AMPK Regulates ATGL-1 Association with Lipid Droplets in Dauer Larvae.(A)-(B) Signal overlap between ATGL-1::GFP (Green) and C1-BODIPY-C12-stained lipid droplets (Red) was compared in control daf-2 and daf-2; aak(0) mutant animals at 32 (A) and 48 hours (B) after shifting to restricted temperature. ATGL-1::GFP signal was closely associated with the labeled lipid droplets in daf-2; aak(0) mutant animals (white arrowheads in the insets) while the signals are clearly distinguishable from each other in control daf-2 animals. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified by the same magnification. (C) Western blot analysis of the endogenous ATGL-1 levels in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 (48 hours after shifting to restricted temperature) dauer extracts of control daf-2 and daf-2; aak(0) mutant animals. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level according to the recent proteomic study on C. elegans lipid droplets [39]. (D) Lipid droplet isolation method verified by significant C1-BODIPY-C12 staining and triglyceride enrichment in the isolated lipid droplets portion comparing to the cytoplasm (remaining portion of the total lysate) from daf-2 day 0 dauer larvae.

Mentions: Given that most triglyceride molecules are stored in the lipid droplets, we next determined whether ATGL-1 associates with the lipid droplets where it can initiate the lipolysis process, and whether this might be under AMPK-mediated regulation. We stained control daf-2 and daf-2; aak(0) dauer larvae that expressed the ATGL-1::GFP translational fusion protein with red C1-BODIPY-C12 to label lipid droplets and subsequently monitored both fluorescent signals 32 and 48 hours after being shifted to restrictive temperature. In control daf-2 dauer larvae, the ATGL-1::GFP signal was sequestered away from the red lipid droplet signal at both the 32 and 48 hour time points, whereas in daf-2; aak(0) dauers the ATGL-1::GFP signal still remained closely associated with the lipid droplets during the later part of the dauer entry period (Fig 6A and 6B).


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)

AMPK Regulates ATGL-1 Association with Lipid Droplets in Dauer Larvae.(A)-(B) Signal overlap between ATGL-1::GFP (Green) and C1-BODIPY-C12-stained lipid droplets (Red) was compared in control daf-2 and daf-2; aak(0) mutant animals at 32 (A) and 48 hours (B) after shifting to restricted temperature. ATGL-1::GFP signal was closely associated with the labeled lipid droplets in daf-2; aak(0) mutant animals (white arrowheads in the insets) while the signals are clearly distinguishable from each other in control daf-2 animals. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified by the same magnification. (C) Western blot analysis of the endogenous ATGL-1 levels in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 (48 hours after shifting to restricted temperature) dauer extracts of control daf-2 and daf-2; aak(0) mutant animals. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level according to the recent proteomic study on C. elegans lipid droplets [39]. (D) Lipid droplet isolation method verified by significant C1-BODIPY-C12 staining and triglyceride enrichment in the isolated lipid droplets portion comparing to the cytoplasm (remaining portion of the total lysate) from daf-2 day 0 dauer larvae.
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Related In: Results  -  Collection

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pone.0130480.g006: AMPK Regulates ATGL-1 Association with Lipid Droplets in Dauer Larvae.(A)-(B) Signal overlap between ATGL-1::GFP (Green) and C1-BODIPY-C12-stained lipid droplets (Red) was compared in control daf-2 and daf-2; aak(0) mutant animals at 32 (A) and 48 hours (B) after shifting to restricted temperature. ATGL-1::GFP signal was closely associated with the labeled lipid droplets in daf-2; aak(0) mutant animals (white arrowheads in the insets) while the signals are clearly distinguishable from each other in control daf-2 animals. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified by the same magnification. (C) Western blot analysis of the endogenous ATGL-1 levels in isolated lipid droplets (L) and cytoplasm (C) obtained from total day 0 (48 hours after shifting to restricted temperature) dauer extracts of control daf-2 and daf-2; aak(0) mutant animals. Protein concentration was measured and 30μg of total protein was loaded in each sample lane. Actin was used as a loading control for the total protein level according to the recent proteomic study on C. elegans lipid droplets [39]. (D) Lipid droplet isolation method verified by significant C1-BODIPY-C12 staining and triglyceride enrichment in the isolated lipid droplets portion comparing to the cytoplasm (remaining portion of the total lysate) from daf-2 day 0 dauer larvae.
Mentions: Given that most triglyceride molecules are stored in the lipid droplets, we next determined whether ATGL-1 associates with the lipid droplets where it can initiate the lipolysis process, and whether this might be under AMPK-mediated regulation. We stained control daf-2 and daf-2; aak(0) dauer larvae that expressed the ATGL-1::GFP translational fusion protein with red C1-BODIPY-C12 to label lipid droplets and subsequently monitored both fluorescent signals 32 and 48 hours after being shifted to restrictive temperature. In control daf-2 dauer larvae, the ATGL-1::GFP signal was sequestered away from the red lipid droplet signal at both the 32 and 48 hour time points, whereas in daf-2; aak(0) dauers the ATGL-1::GFP signal still remained closely associated with the lipid droplets during the later part of the dauer entry period (Fig 6A and 6B).

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.