NHR-49 Helps Germline-Less Worms Chew the Fat

In C. elegans, removal of the germline extends lifespan significantly. We demonstrate that the nuclear hormone receptor, NHR-49, enables the response to this physiological change by increasing the expression of genes involved in mitochondrial β-oxidation and fatty-acid desaturation. The coordinated augmentation of these processes is critical for germline-less animals to maintain their lipid stores and to sustain de novo fat synthesis during adulthood. Following germline ablation, NHR-49 is up-regulated in somatic cells by the conserved longevity determinants DAF-16/FOXO and TCER-1/TCERG1. Accordingly, NHR-49 overexpression in fertile animals extends their lifespan modestly. In fertile adults, nhr-49 expression is DAF-16/FOXO and TCER-1/TCERG1 independent although its depletion causes age-related lipid abnormalities. Our data provide molecular insights into how reproductive stimuli are integrated into global metabolic changes to alter the lifespan of the animal. They suggest that NHR-49 may facilitate the adaptation to loss of reproductive potential through synchronized enhancement of fatty-acid oxidation and desaturation, thus breaking down some fats ordained for reproduction and orchestrating a lipid profile conducive for somatic maintenance and longevity

Targeted lipidomics reveals a novel role for glucosylceramides in glucose response

The addition of excess glucose to the diet drives a coordinated response of lipid metabolism pathways to tune the membrane composition to the altered diet. Here, we have employed targeted lipidomic approaches to quantify the specific changes in the phospholipid and sphingolipid populations that occur in elevated glucose conditions. The lipids within wild-type Caenorhabditis elegans are strikingly stable with no significant changes identified in our global mass spectrometry–based analysis. Previous work has identified ELO-5, an elongase that is critical for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs), as essential for surviving elevated glucose conditions. Therefore, we performed targeted lipidomics on elo-5 RNAi-fed animals and identified several significant changes in these animals in lipid species that contain mmBCFAs as well as in species that do not contain mmBCFAs. Of particular note, we identified a specific glucosylceramide (GlcCer 17:1;O2/22:0;O) that is also significantly upregulated with glucose in wild-type animals. Furthermore, compromising the production of the glucosylceramide pool with elo-3 or cgt-3 RNAi leads to premature death in glucose-fed animals. Taken together, our lipid analysis has expanded the mechanistic understanding of metabolic rewiring with glucose feeding and has identified a new role for the GlcCer 17:1;O2/22:0;O

HPLC-Based Mass Spectrometry Characterizes the Phospholipid Alterations in Ether-Linked Lipid Deficiency Models Following Oxidative Stress

<div><p>Despite the fact that the discovery of ether-linked phospholipids occurred nearly a century ago, many unanswered questions remain concerning these unique lipids. Here, we characterize the ether-linked lipids of the nematode with HPLC-MS/MS and find that more than half of the phosphoethanolamine-containing lipids are ether-linked, a distribution similar to that found in mammalian membranes. To explore the biological role of ether lipids <i>in vivo</i>, we target fatty acyl-CoA reductase (<i>fard-1)</i>, an essential enzyme in ether lipid synthesis, with two distinct RNAi strategies. First, when <i>fard-1</i> RNAi is initiated at the start of development, the treated animals have severely reduced ether lipid abundance, resulting in a shift in the phosphatidylethanolamine lipid population to include more saturated fatty acid chains. Thus, the absence of ether lipids during development drives a significant remodeling of the membrane landscape. A later initiation of <i>fard-1</i> RNAi in adulthood results in a dramatic reduction of new ether lipid synthesis as quantified with ¹⁵N-tracers; however, there is only a slight decrease in total ether lipid abundance with this adult-only <i>fard-1</i> RNAi. The two RNAi strategies permit the examination of synthesis and ether lipid abundance to reveal a relationship between the amount of ether lipids and stress survival. We tested whether these species function as sacrificial antioxidants by directly examining the phospholipid population with HPLC-MS/MS after oxidative stress treatment. While there are significant changes in other phospholipids, including polyunsaturated fatty acid-containing species, we did not find any change in ether-linked lipids, suggesting that the role of ether lipids in stress resistance is not through their general consumption as free radical sinks. Our work shows that the nematode will be a useful model for future interrogation of ether lipid biosynthesis and the characterization of phospholipid changes in various stress conditions.</p></div

Adult-Only <i>fard-1</i> RNAi Treatment Causes Reduced Ether Lipid Production.

<p>(A) ¹⁵N-incorporation assays allowed for the measurement of new phospholipid headgroup incorporation. Phospholipid species are identified here by the summated molecular species that comprise both fatty acid tails. For example O-PE (36:1) represents a plasmanylethanolamine with 36 carbons and one double bond in the associated fatty acids. In <i>fard-1</i> RNAi-treated adults (yellow), there is depletion in newly synthesized O-PE (36:1) and P-PE (36:1), but not PC (36:2) or PE (36:2) as compared to control animals (black). Data was generated from at least 4 independent biological replicates with SEM shown. *p<0.05 was determined by unpaired t-tests using Holm-Sidak corrections for multiple comparisons. (B) Here we present a summary of the lipid biochemical data from developmental and adult-only <i>fard-1</i> RNAi treatments. Developmental <i>fard-1</i> RNAi results in stronger phenotypes across all categories including synthesis where there were not sufficient O-PE and P-PE lipids for analysis.</p

Expression of Ether-Lipid Synthesis Genes is Greatest in Larvae.

<p>In wild-type nematodes, the expression of <i>fard-1</i> is significantly lower in day 1 (grey) adults and day 3 (white) adults when compared to L1 animals (black). A separate analysis was performed to assay the expression of two additional ether lipid biosynthetic genes, <i>acl-7</i> and <i>ads-1</i>. The statistical significance was determined by one-way ANOVA with significant differences (p<0.05) being indicated by *. Data is a result of at least 3 biological replicates with SEM shown.</p

<i>fard-1</i> RNAi Treatments Results in Compromised Ether-Linked Lipid Abundance.

<p>(A) Developmental RNAi-treatment was initiated at the L1 stage and continued until animals were harvested for analysis at day 3 of adulthood, resulting in 4 days of RNAi feeding at 25°C (blue). The second RNAi-treatment described is an adult-only <i>fard-1</i> RNAi strategy (yellow) where RNAi is started after the completion of development at day 1 of adulthood (44 hours post-hatch). Animals were harvested at day 3 for both treatments. (B) qRT-PCR of <i>fard-1</i> expression is reduced to 0.34 ± 0.18 and 0.40 ± 0.17 of wild-type levels with developmental RNAi and adult-only RNAi treatment, respectively. The <i>cdc-42</i> housekeeping gene was used for normalization. (C) When <i>fard-1</i> RNAi is initiated at the L1 stage and continued throughout development until day 3 of adulthood (blue), there is a significant reduction in the relative abundance of both O-PE and P-PE compared to control L4440 RNAi (black). (D) The overall composition of PE was compared in control RNAi- (grey) and <i>fard-1</i> RNAi-treated animals (light blue) after binning the lipids by the overall degree of unsaturation found in the associated fatty acids. Similarly, PC species were measured in RNAi controls (black) and <i>fard-1</i> RNAi-treated animals (dark blue). (E) <i>fard-1</i> RNAi initiated in adult animals at day 1 and maintained until day 3 of adulthood (yellow) resulted in a significant decrease in O-PE as quantified by HPLC-MS/MS. There is a small but not significant decrease in the P-PE population (p-Value of 0.11). (F) The impact of adult-only <i>fard-1</i> RNAi treatment on the PE and PC populations showed no significant changes in the degree of unsaturation in PE (yellow) or PC (orange) compared to L4440 RNAi shown in grey for PE and black for PC. Data was generated from at least 3 independent biological replicates with SEM shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167229#pone.0167229.s006" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167229#pone.0167229.s007" target="_blank">S2</a> Datasets for complete list of phospholipid species. *p<0.05 was determined by unpaired t-tests using Holm-Sidak corrections for multiple comparisons.</p

Elevated Oxidative Stress does not Impact Ether Lipid Abundance.

<p>The abundance (A) and distribution (B) of O-PE and P-PE within the major phospholipids in L4440-fed animals before (grey) and after (black) 100mM PQ treatment is shown. The same comparisons were done in adult-only <i>fard-1</i> treated animals before (yellow) and after (orange) stress treatment. (C) All PC and PE species with significant changes are represented, and the full list is available in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167229#pone.0167229.s008" target="_blank">S3 Dataset</a>. Biochemical analysis was done on at least 12 biological replicates with SEM shown. For simplicity, only changes meeting a statistical significance of p<0.001 are denoted here by ^a for L4440 RNAi and ^b for <i>fard-1</i> RNAi treatment, and the complete statistical analysis is found in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167229#pone.0167229.s008" target="_blank">S3 Dataset</a>. (D) Here, we summarize the changes in the membrane phospholipids after paraquat treatment. The model presented here was generated by considering the major phospholipid species (≥ 2%), thus removing species that may have exhibited significant changes after PQ treatment but compose a very small fraction of the total phospholipids. The PC pool was divided into a group containing ≤7 double bonds and one with ≥8 double bonds as these populations behaved in distinct manners.</p

<i>fard-1</i> is Required for Thermotolerance but not Osmotic Stress in Adult Animals.

<p>(A) A sample of a large population of adult-only <i>fard-1</i> RNAi-treated animals was assessed for viability after 48 hours of 100mM PQ treatment in each population prepared for biochemical analysis. (B) Developmental <i>fard-1</i> RNAi-treated populations were assayed in 100mM PQ as well; however, fewer animals were needed as no HPLC-MS/MS was carried out on those animals. The data presented is normalized to the L4440 controls to account for the differences in culture size. Individual comparisons can be found as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167229#pone.0167229.s005" target="_blank">S5 Fig</a>. (C) Lifespan analysis was completed for developmental <i>fard-1</i> (blue) and adult-only <i>fard-1</i> RNAi treatment (yellow). There was a significant reduction (p<0.001) in the average lifespan with developmental RNAi (13.4 ± 0.8 days) versus control (18.0 ±0.5 days). (D) Viability was assessed for the same <i>fard-1</i> treatments following exposure to 500mM NaCl and (E) after 24 hours of heat stress at 35°C. Data presented here is a result of at least 4 biological replicates containing a minimum of 40 animals per condition in each replicate. SEM is presented, and *p<0.05 was determined by unpaired t-tests using Holm-Sidak corrections for multiple comparisons where applicable.</p

Quantification of Membrane Dynamics With ¹³C-Labeling and GC-MS in Young Adults.

<p>(A) Day 3 adult nematodes were fed a diet of 50% ¹³C-labeled <i>E</i>. <i>coli</i> for 6 hours to introduce ¹³C into their lipids and mark them as newly added or modified. The ¹³C can be traced into the major membrane FAs (>2% abundance) by GC-MS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141850#pone.0141850.s011" target="_blank">S1 Table</a>). The amount of new FA measured by ¹³C-incorporation, was determined for purified PLs (black) and NLs (white). C20s with 4 or more double bonds were excluded in the analysis due to insufficient isotopomers. C20:0 and C20:2 FAs were profiled despite low abundance in the membrane to increase the representation of long-chain FAs. (B) After combining the amount of new FA found in all major C15 to C19 FAs, the overall contribution of new FA/hour was significantly greater in the PLs versus the NLs. (C) The relative amount of new FA derived from <i>de novo</i> FA synthesis was not significantly different for PLs (black) and NLs (white) except for C16:0. Numbers shown represent the mean of at least four experiments ± SEM. Statistical significance was calculated by two-tailed unpaired t-tests where significance (*p<0.05, **p<0.01, ***p<0.001) is noted by the asterisks.</p