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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 Compromise Causes Enlarged Lipid Droplets in C. elegans Dauer Larvae.(A)-(F) Disruption of ATGL-1, but not HSL function in both control daf-2(A), (C) and (E) and daf-2; aak(0)(B), (D) and (F) dauer day 0 animals (48 hours after shifting to restrictive temperature since L1 stage) caused increase in lipid droplet size. Animals were stained with C1-BODIPY-C12. These and all subsequent images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x40 magnification using identical microscope settings, unless specified otherwise. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified to the same magnification. (G)atgl-1(RNAi) feeding reduces endogenous ATGL-1 protein substantially. A Western blot probed with an anti-ATGL-1 polyclonal antibody raised against endogenous ATGL-1 was used to quantify ATGL-1 levels in control daf-2 animals and daf-2 animals subjected to feeding atgl-1(RNAi). (H)-(I) Quantification of the volume (H) and number (I) of C1-BODIPY-C12-stained lipid droplets using AxioVision (Zeiss) software. ** indicates statistical significance (P<0.0001) and ns indicates not significant using the unpaired t test compared to control daf-2 and daf-2; aak(0) animals respectively. Error bars indicate SD of three independent experiments.
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pone.0130480.g001: ATGL-1 Compromise Causes Enlarged Lipid Droplets in C. elegans Dauer Larvae.(A)-(F) Disruption of ATGL-1, but not HSL function in both control daf-2(A), (C) and (E) and daf-2; aak(0)(B), (D) and (F) dauer day 0 animals (48 hours after shifting to restrictive temperature since L1 stage) caused increase in lipid droplet size. Animals were stained with C1-BODIPY-C12. These and all subsequent images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x40 magnification using identical microscope settings, unless specified otherwise. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified to the same magnification. (G)atgl-1(RNAi) feeding reduces endogenous ATGL-1 protein substantially. A Western blot probed with an anti-ATGL-1 polyclonal antibody raised against endogenous ATGL-1 was used to quantify ATGL-1 levels in control daf-2 animals and daf-2 animals subjected to feeding atgl-1(RNAi). (H)-(I) Quantification of the volume (H) and number (I) of C1-BODIPY-C12-stained lipid droplets using AxioVision (Zeiss) software. ** indicates statistical significance (P<0.0001) and ns indicates not significant using the unpaired t test compared to control daf-2 and daf-2; aak(0) animals respectively. Error bars indicate SD of three independent experiments.

Mentions: Given that ATGL-1-deficient dauer larvae were shown to have more triglycerides [9], we first questioned whether the excess triglyceride molecules might have an effect on the lipid droplet size and/or number. Therefore, we stained dauer day 0 (defined as 48 hours after shifting to restrictive temperature at the L1 stage) control daf-2 and daf-2; aak-1; aak-2 (aak-1; aak-2 will be presented as aak(0) hereafter) dauer larvae that were previously fed with regular or atgl-1(RNAi) bacteria using C1-BODIPY-C12 to label their lipid droplets (Fig 1A–1D). The efficiency of our feeding atgl-1(RNAi) protocol was confirmed with a C. elegans ATGL-1-specific antibody (see description below; Fig 1G). We found that following the compromise of atgl-1, the lipid droplet size was significantly increased in both control daf-2 and daf-2; aak(0) dauers (Fig 1H) with no significant changes in lipid droplet number (Fig 1I). This indicates that triglycerides accumulate during the dauer entry phase but because they are not hydrolyzed due to the absence of ATGL-1 they remain encapsulated in the lipid droplets, leading to the enlargement of the organelles. Notably, the removal of hormone sensitive lipase (hosl-1), did not significantly alter the lipid droplet abundance or structure in either control daf-2 or daf-2; aak(0) animals (Fig 1E, 1F and 1H), consistent with its secondary role in lipid hydrolysis during dauer survival [9].


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 Compromise Causes Enlarged Lipid Droplets in C. elegans Dauer Larvae.(A)-(F) Disruption of ATGL-1, but not HSL function in both control daf-2(A), (C) and (E) and daf-2; aak(0)(B), (D) and (F) dauer day 0 animals (48 hours after shifting to restrictive temperature since L1 stage) caused increase in lipid droplet size. Animals were stained with C1-BODIPY-C12. These and all subsequent images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x40 magnification using identical microscope settings, unless specified otherwise. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified to the same magnification. (G)atgl-1(RNAi) feeding reduces endogenous ATGL-1 protein substantially. A Western blot probed with an anti-ATGL-1 polyclonal antibody raised against endogenous ATGL-1 was used to quantify ATGL-1 levels in control daf-2 animals and daf-2 animals subjected to feeding atgl-1(RNAi). (H)-(I) Quantification of the volume (H) and number (I) of C1-BODIPY-C12-stained lipid droplets using AxioVision (Zeiss) software. ** indicates statistical significance (P<0.0001) and ns indicates not significant using the unpaired t test compared to control daf-2 and daf-2; aak(0) animals respectively. Error bars indicate SD of three independent experiments.
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Related In: Results  -  Collection

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pone.0130480.g001: ATGL-1 Compromise Causes Enlarged Lipid Droplets in C. elegans Dauer Larvae.(A)-(F) Disruption of ATGL-1, but not HSL function in both control daf-2(A), (C) and (E) and daf-2; aak(0)(B), (D) and (F) dauer day 0 animals (48 hours after shifting to restrictive temperature since L1 stage) caused increase in lipid droplet size. Animals were stained with C1-BODIPY-C12. These and all subsequent images were taken with a Zeiss 510 Meta Confocal Laser Microscope at x40 magnification using identical microscope settings, unless specified otherwise. Scale bar = 10μm. Insets were generated by selecting the same size of frame on each image and amplified to the same magnification. (G)atgl-1(RNAi) feeding reduces endogenous ATGL-1 protein substantially. A Western blot probed with an anti-ATGL-1 polyclonal antibody raised against endogenous ATGL-1 was used to quantify ATGL-1 levels in control daf-2 animals and daf-2 animals subjected to feeding atgl-1(RNAi). (H)-(I) Quantification of the volume (H) and number (I) of C1-BODIPY-C12-stained lipid droplets using AxioVision (Zeiss) software. ** indicates statistical significance (P<0.0001) and ns indicates not significant using the unpaired t test compared to control daf-2 and daf-2; aak(0) animals respectively. Error bars indicate SD of three independent experiments.
Mentions: Given that ATGL-1-deficient dauer larvae were shown to have more triglycerides [9], we first questioned whether the excess triglyceride molecules might have an effect on the lipid droplet size and/or number. Therefore, we stained dauer day 0 (defined as 48 hours after shifting to restrictive temperature at the L1 stage) control daf-2 and daf-2; aak-1; aak-2 (aak-1; aak-2 will be presented as aak(0) hereafter) dauer larvae that were previously fed with regular or atgl-1(RNAi) bacteria using C1-BODIPY-C12 to label their lipid droplets (Fig 1A–1D). The efficiency of our feeding atgl-1(RNAi) protocol was confirmed with a C. elegans ATGL-1-specific antibody (see description below; Fig 1G). We found that following the compromise of atgl-1, the lipid droplet size was significantly increased in both control daf-2 and daf-2; aak(0) dauers (Fig 1H) with no significant changes in lipid droplet number (Fig 1I). This indicates that triglycerides accumulate during the dauer entry phase but because they are not hydrolyzed due to the absence of ATGL-1 they remain encapsulated in the lipid droplets, leading to the enlargement of the organelles. Notably, the removal of hormone sensitive lipase (hosl-1), did not significantly alter the lipid droplet abundance or structure in either control daf-2 or daf-2; aak(0) animals (Fig 1E, 1F and 1H), consistent with its secondary role in lipid hydrolysis during dauer survival [9].

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.