Sphingolipid metabolism regulates development and lifespan in Caenorhabditis elegans

Abstract

Sphingolipids are a highly conserved lipid component of cell membranes involved in the formation of lipid raft domains that house many of the receptors and cell-to-cell signaling factors involved in regulating cell division, maturation, and terminal differentiation. By measuring and manipulating sphingolipid metabolism using pharmacological and genetic tools in Caenorhabditis elegans, we provide evidence that the synthesis and remodeling of specific ceramides (e.g., dC18:1-C24:1), gangliosides (e.g., GM1-C24:1), and sphingomyelins (e.g., dC18:1-C18:1) influence development rate and lifespan. We found that the levels of fatty acid chain desaturation and elongation in many sphingolipid species increased during development and aging, with no such changes in developmentally-arrested dauer larvae or normal adults after food withdrawal (an anti-aging intervention). Pharmacological inhibitors and small interfering RNAs directed against serine palmitoyl transferase and glucosylceramide synthase acted to slow development rate, extend the reproductive period, and increase lifespan. In contrast, worms fed an egg yolk diet rich in sphingolipids exhibited accelerated development and reduced lifespan. Our findings demonstrate that sphingolipid accumulation and remodeling are critical events that determine development rate and lifespan in the nematode model, with both development rate and aging being accelerated by the synthesis of sphingomyelin, and its metabolism to ceramides and gangliosides.

Keywords: Ceramide; Gangliosides; Longevity; Oxidative stress; Sphingomyelin.

Fig. 1

Sphingolipid metabolic pathways and shotgun lipidomic analysis of aging and dauer arrested C. elegans. (A) Biochemical pathways for the synthesis and metabolism of ceramides, gangliosides, and sphingomyelins. Drug- and RNAi-targeted enzymes evaluated in this study are indicated. (B) Representative tandem mass spectrograms showing Q1 detection of all lipid species from 150 to 2000 Daltons [m/z (mass/charge), amu (atomic mass units)] in samples from adult C. elegans of the indicated ages, and 3 day old dauer larvae. Note the increasing amounts of gangliosides GM1 and GM3 in adults compared to dauers, and with advancing age in adults.

Fig. 2

Levels of unsaturated ceramides, and gangliosides GM1 and GM3, increase with advancing age. Results of quantitative analysis of the indicated dihydroceramides (A), ceramides (B) and gangliosides (C) and (D) in C. elegans eggs, dauer larvae, and adults of the indicated ages. Values are the mean and SEM (n = 4 separate experiments). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the level of the same lipid in 3 day-old adult worms.

Fig. 3

Levels of sphingomyelins and triglycerides change during development and aging. Results of quantitative analysis of levels of the indicated dihydrosphingomyelins (A), sphingomyelins (B) and triglycerides (C) in C. elegans eggs, dauer larvae, and adults of the indicated ages. Values are the mean and SEM (n = 4 separate experiments). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the level of the same lipid in 3 day-old adult worms.

Fig. 4

Development rate is slowed in worms treated with an inhibitor of de novo sphingolipid synthesis. (A) Plots show development rates of C. elegans maintained at the indicated temperatures, or treated with ISP-1 (at 20 ° C). **p < 0.01 and ***p < 0.001 compared to untreated worms maintained at 20 ° C. B. The duration of the egg laying period, a measure of functional aging, is significantly increased in worms treated with ISP-1 at 20 ° C (p < 0.01).

Fig. 5

The accumulation of oxidatively damaged lipids is attenuated in worms treated with an inhibitor of de novo sphingolipid synthesis. (A) Examples of auto-fluorescence in worms of the indicated ages. (B) Results of quantitative measurements of auto-fluorescence in worms of different ages. (C) The age-related accumulation of auto-fluorescent/oxidized lipids is attenuated in worms treated with the SPT inhibitor ISP-1. **p < 0.01 compared to worms not treated with ISP-1.

Fig. 6

Pharmacological and genetic inhibition of sphingolipid metabolism results in an extension of lifespan. (A) Lifespan was determined in worms maintained in the presence of chemical inhibitors of the indicated enzymes (triacylglycerol synthase, C75; serine palmitoyl transferase, ISP-1; dihydroceramide desaturase, C8-CPC; ceramidase, MAPP; glucosylceramide synthase, PDMP; sphingomyelin synthase, D609; neutral sphingomyelinase, epoxyquinon G109). Lifespan was significantly extended in worms treated with inhibitors of serine palmitoyl transferase, glucosylceramide synthase, and sphingomyelin synthase (p < 0.01). (B) The effects of genetic siRNA targeting of enzymes involved in sphingolipid metabolism on lifespan was determined in worms in which the indicated enzymes were targeted with siRNAs. Lifespan was significantly extended in worms in which serine palmitoyl transferase, dihydroceramide desaturase, ceramidase or glucosylceramide synthase were targeted (p < 0.01). In contrast, lifespan was shortened when neutral sphingomeylinase was targeted (p < 0.01). (C) Serine palmitoyl transferase 1 (sptl-1) mutant worms exhibit a significant extension of lifespan (p < 0.01).