ATGL has a key role in lipid droplet/adiposome degradation in mammalian cells

Abstract

Lipid droplets (LDs), also called adiposomes, are found in many eukaryotic cells, and are highly upregulated in lipid-storage cells, such as adipocytes. The mechanism by which adiposomes and their component neutral lipids are degraded is an important health issue with the rapidly spreading epidemic of obesity. Recently, a novel triglyceride lipase (adipose triglyceride lipase (ATGL)) that catalyses the initial step in triglyceride hydrolysis in adipocyte LDs was identified. Here, we show that ATGL also functions in non-adipocyte cells, and has an important role in LD degradation in these cells. Overexpression of wild-type ATGL causes a marked decrease in LD size, whereas a catalytically inactive mutant retains the ability to localize to LDs, but is unable to decrease their size. Depletion of ATGL by RNA interference leads to a significant increase in the size of LDs. These results show that ATGL has an important role in LD/adiposome turnover in mammalian cells.

Figure 1

Endogenous ATGL localizes to adiposomes. HeLa cells were immunostained with antibodies against ATGL and either anti-TIP47 antibodies or the Nile Red (or BODIPY 493/503) dye. Cells were incubated overnight with 62.5 μM oleic acid complexed to albumin (OA/BSA), where indicated. (A) Cells grown in normal low-lipid-containing medium show colocalization of ATGL (green) with TIP47 (red; upper panel) and colocalization of ATGL (green) with Nile Red (red; lower panel). (B) Cells grown overnight in the presence of medium supplemented with 62.5 μM OA/BSA show colocalization of ATGL (green) with TIP47 (red; upper panel) and ATGL (red) with BODIPY 493/503 dye (green; lower panel).

Figure 2

Overexpression of ATGL causes a decrease in the size of adiposomes. HeLa cells were transiently transfected with plasmid expressing ATGL–GFP (green fluorescent protein) or ATGL-S47A–GFP and analysed by immunofluorescence 24 h after transfection. (A) ATGL–GFP (green) and TIP47 (red) in cells growing under normal conditions (upper panel), or incubated with 400 μM oleic acid complexed to albumin (OA/BSA; lower panel). (B) Sequence alignment of a portion of the catalytic domain of ATGL and related lipases showing the catalytic serine (^*; for details, see supplementary Fig S1 online). (C) ATGL-S47A–GFP (green) and TIP47 (red) in cells growing under regular conditions (upper panel), or incubated with 400 μM OA/BSA (lower panel). (D) Quantification of the results shown in (A,C). Data shown are mean±standard deviation. An average of 120 lipid droplets (LDs) was measured for each point. WT, wild type.

Figure 3

Depletion of ATGL by RNA interference increases the size of lipid droplets. (A) HeLa cells were treated with ATGL short interfering RNA (siRNA; lower panel) or control unrelated siRNA (upper panel) for 72 h, and were then analysed by immunofluorescence microscopy. Overlay images are shown in the left column, staining for endogenous ATGL (green) is in the centre and TIP47 (red) is on the right. (B) Same as (A), except that cells were incubated with 400 μM oleic acid complexed to BSA before fixation. (C) Immunoblot analysis of ATGL expression. HeLa cells were transfected with control siRNA (lanes 1,2) or ATGL siRNA (lanes 3,4). siRNAs were transfected either alone (lanes 1,3) or in combination with a plasmid expressing ATGL–GFP (green fluorescent protein; lanes 2,4). ATGL–GFP was detected using an anti-ATGL antibody. Tubulin and actin were used as loading controls. (D) Quantification of the results shown in (A,B). Data shown are mean±standard deviation. An average of 350 lipid droplets (LDs) was measured for each point.

Figure 4

Phylogenetic tree for lipases of the patatin group. Maximum-likelihood unrooted tree was built using the MOLPHY program. The same program was used to compute bootstrap probabilities. Each terminal node of the tree is labelled by the scientific name of the organism in which the protein is encoded and the numeric GenBank identifier (GI). The names for experimentally characterized proteins are indicated in parentheses and are shown in bold type. Those main branches that were supported by bootstrap probability greater than 70% are marked by black circle. Principal protein families are named by a typical representative and by a KOG/COG number from the database at NCBI (http://www.ncbi.nlm.nih.gov/COG/new/). Branches and nodes for bacterial sequences are highlighted in magenta and those for eukaryotic sequences in green.

Figure 5

Yeast Tgl3p expressed in HeLa cells localizes to lipid droplets. HeLa cells were transfected with a plasmid encoding Saccharomyces cerevisiae Tgl3p fused to green fluorescent protein (GFP). At 24 h after transfections, cells were immunostained with TIP47 antibodies (red). Tgl3p–GFP fluorescence is in green. Two separate microscopy fields are shown in the upper and lower panels.