Regulation of C. elegans fat uptake and storage by acyl-CoA synthase-3 is dependent on NR5A family nuclear hormone receptor nhr-25

Abstract

Acyl-CoA synthases are important for lipid synthesis and breakdown, generation of signaling molecules, and lipid modification of proteins, highlighting the challenge of understanding metabolic pathways within intact organisms. From a C. elegans mutagenesis screen, we found that loss of ACS-3, a long-chain acyl-CoA synthase, causes enhanced intestinal lipid uptake, de novo fat synthesis, and accumulation of enlarged, neutral lipid-rich intestinal depots. Here, we show that ACS-3 functions in seam cells, epidermal cells anatomically distinct from sites of fat uptake and storage, and that acs-3 mutant phenotypes require the nuclear hormone receptor NHR-25, a key regulator of C. elegans molting. Our findings suggest that ACS-3-derived long-chain fatty acyl-CoAs, perhaps incorporated into complex ligands such as phosphoinositides, modulate NHR-25 function, which in turn regulates an endocrine program of lipid uptake and synthesis. These results reveal a link between acyl-CoA synthase function and an NR5A family nuclear receptor in C. elegans.

Figure 1. Mutation of acs-3, a long chain acyl-CoA synthase, results in altered fat storage

From a mutagenesis screen we identified a line, acs-3(ft5), that exhibits elevated staining when fed bacteria labeled both with Nile Red (50nM) and BODIPY-labeled fatty acid (A). Further analysis of acs-3(ft5) mutants revealed the presence of large refractile droplets visible using DIC microscopy, which were not seen in wild type animals. These large droplets stain with BODIPY-labeled fatty acid (B). The lipase ATGL, a lipid droplet associated protein, appears as rings associated with the perimeter of these large droplets in acs-3(ft5) mutant animals (C). When acs-3(ft5) mutant animals are labeled with 5μM (100x) Nile Red, these droplets stain yellow-gold, while intestinal cells of wild type animals lack these large droplets (D). To measure emission spectra, we utilized a spectral confocal microscope, exciting with a 488nm laser, and collecting emitted light at 5nm intervals between 500nm and 660nm. The emission peak for an individual droplet is between 470-480nm, indicating that these large droplets contain neutral lipids (E). When fixed and stained with the lipid dye Sudan Black, these large droplets are clearly labeled (F). In all panels, arrows indicate the position of abnormally large lipid droplets. In (C), the scale bar represents 5μM and N denotes the position of an intestinal cell nucleus.

Figure 2. acs-3 has a limited expression pattern, and expression of acs-3 in seam cells rescues the fat storage phenotype

To examine the expression pattern of acs-3, we fused 2.5kb of upstream regulatory sequence to GFP. This construct yields high expression in a limited set of tissues. In L4 animals, strong expression is evident in seam cells, which are beginning to fuse at this stage, the excretory cell (marked Ex) and vulva (marked Vul) as well as cells in the head and tail (A). In an L2 animal, individual seam cells are clearly visible (B). We also generated constructs driving the acs-3 cDNA fused to GFP. We drove this construct with the previously characterized seam cell promoters wrt-2 (C) and grd-10 (D). With both constructs, ACS-3 is clearly localized to the cell membrane (C-D). When we drove the acs-3(ft5) mutant cDNA we observed misslocalization of the protein to seam cell nuclei (E). In panels A-E, arrows mark the position of seam cells. Elevated Nile Red staining of acs-3(ft5) animals can be rescued by expression of acs-3 cDNA with regulatory elements upstream of acs-3 start site, as well as with the wrt-2 and grd-10 seam cell specific promoters (E). Expression in the body-wall muscle, a tissue in which acs-3 is not normally expressed, driven by the myo-3 promoter, fails to rescue the high Nile Red staining of acs-3(ft5) animals (F). We quantified Nile Red intensity in these lines (G). Data are shown as a percentage of the wild-type average +/− SEM. ***p < 0.001 compared to acs-3(ft5).

Figure 3. Characterization of metabolic parameters reveals acs-3(ft5) animals have an increased rate of fatty acid uptake and elevated de novo fatty acid synthesis

We examined a number of physiological parameters that could impact lipid storage. To assess β-oxidation, we incubated animals in media containing tritiated fatty acids, collected that media and measured amount of tritiated water produced (see methods for more details). We found that acs-3(ft5) animals exhibited an increased rate of β-oxidation (A). Measures of pharyngeal pumping rate and progeny production revealed no differences between wild type and acs-3(ft5) mutants (B-C). To examine fatty acid uptake, we incubated animals in media containing BODIPY-labeled fatty acids for 20 minutes, then measured fluorescence intensity. We found that acs-3(ft5) mutants exhibit more than double the rate of fatty acid uptake observed in wild type animals (D). For panels A-D, data are displayed as a percentage of the wild-type average +/− SEM. ***p < 0.001 compared to wild-type. Using 13C labeling, we found that acs-3(ft5) mutants exhibit a higher percentage of de novo synthesized fatty acids (E). **p<0.01, ***p < 0.001 compared to wild type. GC/MS analysis reveals no substantial changes in fatty acid composition between acs-3(ft5) mutants and wild type animals (F). Data are shown as a percentage of total fatty acid +/− SEM.

Figure 4. An unbiased mutagenesis screen identifies two acyl-CoA dehydrogenases and a fatty acyl-CoA elongase as suppressors of acs-3(ft5) growth and fat storage phenotypes

acs-3(ft5) animals exhibit a developmental arrest when treated with the PI-3 kinase inhibitor LY294002. We performed an unbiased mutagenesis screen utilizing this phenotype, and identified two alleles of acdh-11, one allele of acdh-10 and one allele of elo-6 as suppressors of this developmental phenotype (A). These mutations also suppressed the high fatty acid uptake of acs-3(ft5) mutants, but do not affect uptake in an otherwise wild type background (B). The elevated Nile Red staining phenotype of acs-3(ft5) mutants was also suppressed by these mutations, while each of these suppressors, when outcrossed to an otherwise wild type background exhibited normal Nile Red staining phenotype (C-D). Data are displayed as a percentage of the wild-type average +/− SEM. **p < 0.01, ***p < 0.001 compared to acs-3(ft5).

Figure 5. Mutation of nhr-25 suppresses the high Nile Red staining and fatty acid uptake phenotypes of acs-3(ft5) animals

The hypomorphic nhr-25(ku217) allele partially suppresses the developmental arrest of acs-3(ft5) mutant animals grown on LY294002 (A). Arrow indicates position of an arrested acs-3(ft5) animal. nhr-25(ku217) also suppresses the high fatty acid uptake phenotype and high Nile Red staining phenotype of acs-3(ft5) animals, but nhr-25(ku217) in an otherwise wild type background exhibits wild type fatty acid uptake and Nile Red staining (B-D). Data are shown as a percentage of the wild-type average +/− SEM. ***p < 0.001 compared to acs-3(ft5). We also tested the affect of acs-3(ft5) on previously described nhr-25(ku217) phenotypes. We found that loss of acs-3 function can partially suppress both the sterility and egg-laying phenotypes of nhr-25(ku217) (E-F). Data are shown as a percent of all animals assayed. Because the severity of the egg-laying and sterility phenotypes in nhr-25(ku217) is temperature dependant, we performed these assays at 24 degrees, a temperature at which these phenotypes are strong, but most animals develop to adulthood.

Figure 6. NHR-25 ligand-binding domain binds phosphoinositides

Mammalian SF-1, an nhr-25 homolog, has been shown to bind phosphoinositide species. To test the ability of NHR-25 to bind these lipids, we generated and purified recombinant NHR-25 ligand binding domain. Incubation of this protein with PI(4,5)P and PI(3,4,5)P alters retention of the protein in ion exchange chromatography (A) indicating the protein has bound the lipid. Incubation of each phohsphoinositide species with NHR-25 ligand binding domain protein induced ~50% of the protein to bind lipid (B).