Lipid droplet protein LID-1 mediates ATGL-1-dependent lipolysis during fasting in Caenorhabditis elegans

Abstract

Lipolysis is a delicate process involving complex signaling cascades and sequential enzymatic activations. In Caenorhabditis elegans, fasting induces various physiological changes, including a dramatic decrease in lipid contents through lipolysis. Interestingly, C. elegans lacks perilipin family genes which play a crucial role in the regulation of lipid homeostasis in other species. Here, we demonstrate that in the intestinal cells of C. elegans, a newly identified protein, lipid droplet protein 1 (C25A1.12; LID-1), modulates lipolysis by binding to adipose triglyceride lipase 1 (C05D11.7; ATGL-1) during nutritional deprivation. In fasted worms, lipid droplets were decreased in intestinal cells, whereas suppression of ATGL-1 via RNA interference (RNAi) resulted in retention of stored lipid droplets. Overexpression of ATGL-1 markedly decreased lipid droplets, whereas depletion of LID-1 via RNAi prevented the effect of overexpressed ATGL-1 on lipolysis. In adult worms, short-term fasting increased cyclic AMP (cAMP) levels, which activated protein kinase A (PKA) to stimulate lipolysis via ATGL-1 and LID-1. Moreover, ATGL-1 protein stability and LID-1 binding were augmented by PKA activation, eventually leading to increased lipolysis. These data suggest the importance of the concerted action of lipase and lipid droplet protein in the response to fasting signals via PKA to maintain lipid homeostasis.

FIG 1

ATGL-1 is a major lipase for fasting-induced lipolysis in C. elegans. (A) RNA interference (RNAi) screening of lipases involved in fasting-induced lipolysis based on Oil Red O staining. Oil Red O staining intensities of young adult worms in the RNAi groups under feeding and 8-h-fasting conditions were quantified and classified according to relative fold increase compared to the L4440 control. (B and C) Representative images and quantitation data of Oil Red O staining with or without atgl-1 RNAi in young adult worms under feeding and 8-h-fasting conditions. Marked areas were subjected to quantitation of Oil Red O staining. (D) Relative triglyceride amounts of young adult worms were measured with a biochemical triglyceride assay kit and normalized by total worm extract protein. (E) Confocal microscopic image of atgl-1(hj67) worms after fixation and Nile red staining. The inset is a magnified view. (F) mRNA levels of atgl-1 and fasting-responsive genes (fil-1, cpt-2, acs-2, and fat-7) measured by qRT-PCR and normalized to act-1/3 mRNA. (G) Confocal microscopic images of atgl-1(hj67) worms under feeding and fasting conditions at the young adult stage. Scale bars, 10 μm. White lines indicate the boundaries of worm bodies. Error bars represent standard deviations. *, P < 0.05; **, P < 0.01. N.S., not significant.

FIG 2

LID-1 is a homolog of CGI-58 and localizes to lipid droplets. (A) Phylogenic trees of CGI-58 gene family members on the basis of amino acid sequences from Homo sapiens (Hs), Mus musculus (Mm), Danio rerio (Dr), and Caenorhabditis elegans (Ce). Amino acid sequences encoded by all genes were subjected to multiple alignments and analyzed for phylogenetic trees obtained by the neighbor-joining method using ClustalW. (B) Alignment of the GXSXG motif in the α/β hydrolase domain of CGI-58 gene family. (C) Homology scores of LID-1 (C25A1.12) obtained from the Homologene database. (D) Confocal images showing the localization of GFP-fused LID-1 in live worms and in fixed worms costained with Oil Red O and Nile red. The stained area of the Oil Red O image is pseudocolored red. The insets are magnified views. Scale bars, 10 μm. White lines indicate the boundaries of worm bodies. (E) Lysophosphatidyl choline acyltransferase activity assay. One-microgram quantities of total bacterial protein and purified recombinant proteins were used.

FIG 3

LID-1, a C. elegans homolog of CGI-58, is required for ATGL-1 activity. (A and B) Representative images and quantitation data of Oil Red O staining with or without lid-1 RNAi in young adult worms under feeding or fasting conditions. Marked areas were subjected to quantitation of Oil Red O staining. (C and D) Representative images and quantitation data of Oil Red O staining in atgl-1(hj67) worms after L4440 control and lid-1 RNAi. Marked areas were subjected to quantitation of Oil Red O staining. (E) Coimmunoprecipitation (IP) assay of HEK293T cells expressing ATGL-1-Flag and LID-1-Myc. (F) Confocal images of atgl-1(hj67) worms under feeding and fasting conditions after L4440 control or lid-1 RNAi. Scale bars, 10 μm. White lines indicate the boundaries of worm bodies. Error bars represent standard deviations. **, P < 0.01.

FIG 4

PKA signaling mediates fat mobilization upon fasting in C. elegans. (A and B) Representative images and quantitation data of Oil Red O staining with or without kin-1 RNAi in young adult worms under feeding or fasting conditions. (C) Cyclic AMP (cAMP) concentrations measured by direct ELISA in total extracts of wild-type worms under feeding and 4-h-fasting conditions. (D and E) Representative images and quantitation data of Oil Red O staining in wild-type and AMPK mutant worms under feeding or fasting conditions. (F and G) Representative images and quantitation of Oil Red O intensities with control, aak-1, or aak-2 RNAi in young adult worms under feeding or fasting conditions. (H and I) Representative images and quantitation data of Oil Red O staining in the PKA hyperactive kin-2 mutant strain (ce179 strain) after RNAi of atgl-1 or lid-1. For Oil Red O staining, marked areas were subjected to quantitation. Error bars represent standard deviations. *, P < 0.05; **, P < 0.01.

FIG 5

PKA phosphorylates and regulates ATGL-1 functions. (A) Potential PKA phosphorylation site prediction results obtained using the GPS2.1 software were sorted according to the scores indicated. (B) In vitro kinase assay with wild-type (WT) ATGL-1 and S303A ATGL-1 recombinant proteins. Purified GST-fused ATGL-1 proteins were incubated with the PKA catalytic subunit and ³²P-labeled ATP before SDS-PAGE. (C) Confocal images of atgl-1(hj67) worms after L4440 control and kin-2 RNAi. (D and E) Representative images and quantitation of ATGL-1::GFP intensities of atgl-1(hj67) worms under control or forskolin (FSK; 100 μM) treatment. (F) Quantitation of Western blots of C. elegans ATGL-1 proteins in Cos-1 cells with or without forskolin (50 μM, 3 h) treatment. (G) Western blot analysis of mammalian ATGL protein levels in differentiated 3T3-L1 adipocytes treated with forskolin (20 mM) for the indicated time periods. The GAPDH protein was used as a loading control. Scale bars, 20 μm. White lines indicate the boundaries of worm bodies. Error bars represent standard deviations. **, P < 0.01.

FIG 6

PKA increases ATGL-1 protein stability and LID-1 binding. (A) Western blot data of C. elegans ATGL-1 proteins in Cos-1 cells treated with cycloheximide (CHX; 50 μg/ml). Forskolin (50 μM) was pretreated for 3 h. GAPDH was used as a loading control. (B) Confocal images of atgl-1(hj67) worms treated with DMSO, forskolin (100 μM), or MG132 (100 μM) under feeding or fasting conditions. (C) Ubiquitination assay of ATGL-1 in Cos-1 cells. (D) Coimmunoprecipitation assay of Cos-1 cells expressing ATGL-1-Flag and LID-1-Myc in the absence or presence of forskolin (50 μM, 3 h). (E) Confocal images of atgl-1(hj67) worms after L4440 control and kin-1 RNAi under feeding or 4-h-fasting conditions. Scale bars, 20 μm. White lines indicate the boundaries of worm bodies.

FIG 7

atgl-1 and lid-1 are required for energy production during fasting. (A) Oxygen consumption rates of worms in oxygen-saturated M9 buffer after feeding and fasting, normalized to the amounts of total proteins. For details, see Materials and Methods. (B) mRNA levels of cpt-3 measured by qRT-PCR and normalized to act-1/3 mRNA. Error bars represent standard deviations. **, P < 0.01.

FIG 8

Working model. In the feeding state, ATGL-1 is actively degraded by the proteasome pathway to maintain a low rate of basal lipolysis. In the fasting state, increased cAMP levels activate PKA signaling. Then, PKA phosphorylates and stabilizes ATGL-1, which is recruited to lipid droplets via interaction with LID-1, eventually stimulating lipid hydrolysis.