Aberrant fat metabolism in Caenorhabditis elegans mutants with defects in the defecation motor program

Abstract

The molecular mechanisms by which dietary fatty acids are absorbed by the intestine, and the way in which the process is regulated are poorly understood. In a genetic screen for mutations affecting fat accumulation in the intestine of Caenorhabditis elegans, nematode worms, we have isolated mutations in the aex-5 gene, which encodes a Kex2/subtilisin-family, Ca2+-sensitive proprotein convertase known to be required for maturation of certain neuropeptides, and for a discrete step in an ultradian rhythmic phenomenon called the defecation motor program. We demonstrate that aex-5 mutants have markedly lower steady-state levels of fat in the intestine, and that this defect is associated with a significant reduction in the rate at which labeled fatty acid derivatives are taken up from the intestinal lumen. Other mutations affecting the defecation motor program also affect steady-state levels of triglycerides, suggesting that the program is required per se for the proper accumulation of neutral lipids. Our results suggest that an important function of the defecation motor program in C. elegans is to promote the uptake of an important class of dietary nutrients. They also imply that modulation of the program might be one way in which worms adjust nutrient uptake in response to altered metabolic status.

Fig 1. sv75 and sv76 cause decreased fat accumulation in the intestine.

Representative micrographs of parts of young adult hermaphrodite worms viewed with differential interference contrast (DIC) (A-L) or fluorescence (M-O) optics. Scale bars are 10 μm. A-F show parts of the intestines of living worms; G-I show fixed worms stained with the lipophilic dye, Sudan Black. J-L show fixed worms stained with the lipophilic dye, Oil Red O. M-O show fixed worms stained with the fluorescent lipophilic dye, Nile Red. The arrow in D indicates a lipid droplet. P shows quantification (in arbitrary units) of the combined volume of Nile Red-stained droplets relative to tissue volume in wild type and in the mutants (Materials and Methods). Error bars indicate standard errors of the means. * and ** indicate significant differences between the means determined by one-way ANOVA (σ = 0.05) and Dunnett's multiple comparisons test.

Fig 2. sv75 and sv76 mutant worms have lower triglyceride/phospholipid ratios than the N2 wild-type strain.

The graph shows normalised ratios of total triglycerides (TG) to total phospholipids (PL) in extracts from young adult hermaphrodites. Levels of triglycerides and phospholipids relative to C13:0 and C17:0 standards respectively were determined by gas-liquid chromatography. Three extracts were examined for each genotype. Error bars represent 95% confidence intervals. * Indicates significant difference in the means determined by one-way ANOVA and Bonferroni test for differences between means (σ = 0.05).

Fig 3. sv75 and sv76 are alleles of aex-5.

A. Micrographs of fixed young adult hermaphrodites stained with Oil Red O (top panels) or Sudan Black (bottom panels). In wild-type (N2) worms, the dyes stain the intestine and the oocytes within the gonad. In the aex-5(sa23) mutant, only the oocytes show significant staining. Scale bars are 10 μm. B. Graph showing triglyceride/phospholipid ratios determined by gas-liquid chromatography. Error bars represent 95% confidence intervals. * Indicates significant difference in the means determined by one-way ANOVA and Fischer's test for least significant difference (σ = 0.05). C. Schematic diagram of the aex-5 gene (at top) and protein (at bottom). White boxes and lines at top represent exons and introns respectively. The positions of the codons and amino acids affected by the sv75 and sv76 mutations are marked.

Fig 4. Fluorescence confocal micrographs of the intestines of young adult worms stained with Nile Red in isopropanol.

The worms were subjected to RNAi by feeding after they had become adults. L4440 is the negative control in which wild-type adult worms were fed bacteria containing the plasmid vector L4440 alone. The aex-5(RNAi) worms were fed bacteria expressing double-stranded RNA from part of the aex-5 gene.

Fig 5. aex-5 mutants take up a fluorescently labelled fatty acid derivative from the intestinal lumen less efficiently than wild type.

A. Confocal fluorescence micrographs of worms fed the fluorescently labelled fatty acid derivative C₁-BODIPY 500/510 C₁₂. The panels at the top show parts of the intestines of adult worms; those at the bottom show first larval stage (L1) worms. The arrows point to the intestinal lumen where most of the dye is found in the mutants. Scale bars are 20 μ m. B. Graphs showing quantification (in arbitrary units) of the uptake of the dye into the cytoplasm of intestinal cells (Materials and Methods). Error bars indicate standard errors of the means. **** indicates significant difference in the means determined by one-way ANOVA and Dunnett's multiple comparisons test (σ = 0.05).

Fig 6. Mutations in aex-1 and aex-2 also cause a reduction in fat accumulation.

A-C. Fixed, young adult hermaphrodite worms stained with Oil Red O. D-F. Micrographs showing part of the intestines of fixed, young adult hermaphrodite worms stained with Sudan Black. The staining in E and F is of eggs within the gonad. G-I. Fluorescence confocal micrographs of parts of young adult hermaphrodite worms fixed with isopropanol and stained with Nile Red. J. Graph showing the normalized ratios of total triglycerides to total phospholipids extracted from young adult hermaphrodite worms. Error bars represent 95% confidence intervals. * Indicates significant difference in the means determined by one-way ANOVA and Fischer's test for least significant difference (σ = 0.05).

Fig 7. Other defecation mutants also display reduced uptake of C₁-BODIPY 500/510 C₁₂.

A. Confocal fluorescence micrographs of worms fed the fluorescently labelled fatty acid derivative C₁-BODIPY 500/510 C₁₂. The arrows indicate the lumens of the intestine. In all panels, the anterior part of the intestine is up and (in most) slightly to the right. Scale bars are 40 μm. B. Quantification of the uptake of the dye into the cytoplasm of intestinal cells (in arbitrary units). Error bars indicate standard errors of the means. **** indicates significant difference in the means determined by one-way ANOVA and Dunnett's multiple comparisons test (σ = 0.05).