Novel Insight into the Serum Sphingolipid Fingerprint Characterizing Longevity

Abstract

Sphingolipids (SLs) are structural components of the lipid bilayer regulating cell functions. In biological fluids, their distribution is sex-specific and is at variance in aging and many disorders. The aim of this study is to identify SL species associated with the decelerated aging of centenarians. SLs, extracted from serum of adults (Ad, 35-37 years old), aged (Ag, 75-77 years old) and centenarian (C, 105-107 years old) women were analyzed by LC-MS/MS in combination with mRNA levels in peripheral blood mononuclear cells (PBMCs) of SL biosynthetic enzymes. Results indicated in Ag and C vs. Ad a comparable ceramides (Cers) increase, whereas dihydroceramide (dhCer) decreased in C vs. Ad. Hexosylceramides (HexCer) species, specifically HexCer 16:0, 22:0 and 24:1 acyl chains, increased in C vs. Ag representing a specific trait of C. Sphingosine (Sph), dihydrosphingosine (dhSph), sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), increased both in Ag and C vs. Ad, with higher levels in Ag, indicating a SL fine-tuning associated with a reduced physiological decline in C. mRNA levels of enzymes involved in ceramide de novo biosynthesis increased in Ag whereas enzymes involved in sphingomyelin (SM) degradation increased in C. Collectively, results suggest that Ag produce Cers by de novo synthesis whereas C activate a protective mechanism degrading SMs to Cers converting it into glycosphingolipids.

Keywords: ROS; aging; centenarians; longevity; mass spectrometry; nitric oxide; sphingolipids.

Figure 1

Untargeted MS analysis results for Cers (A) and dhCer d18:0/24:0 (B). Results are expressed as pmol/mL in Log scale (Ad n. 15, Ag n.15, C n.15). p-values are indicated as: * p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001.

Figure 2

Untargeted MS analysis results for SMs (A) and for dihydrosphingomyelins (dhSMs) (B). Results are expressed as pmol/mL in Log scale (Ad n. 15, Ag n.15, C n.15). p-values are indicated as: * p-value < 0.05 and ** p-value < 0.01.

Figure 3

Untargeted MS analysis results for HexCers. Results are expressed as pmol/mL in Log scale (Ad n. 15, Ag n.15, C n.15). Total value is expressed as the sum of all quantified acyl chains of the selected sphingolipid. p-values are indicated as: * p-value < 0.05, ** p-value < 0.01 and *** p-value < 0.001.

Figure 4

Untargeted MS analysis results for diHexCer d18:1/24:0 and GM3 d18:1/24:1. Results are expressed as pmol/mL in Log scale (Ad n. 15, Ag n.15, C n.15). p-values is indicated as: * p-value < 0.05.

Figure 5

Targeted MS analysis results for Sph (A), dhSph (B), S1P (C) and dhS1P (D). Results are expressed as pmol/mL in Log scale (Ad n. 15, Ag n.15, C n.15). p-values are indicated as: ** p-value < 0.01 and *** p-value < 0.001.

Figure 6

mRNA levels for SPTLC1 and 2 (A), DEGS1 (B), SMPD3 (C) and UGCG (D) (Ag n.91, C n. 30). p-values is indicated as: *** p-value < 0.001.

Figure 7

UGCG protein expression in PBMC from immunoblotting. p-values are indicated as: ** p-value < 0.01 and *** p-value < 0.001.

Figure 8

Nitro/oxidative state in aged and centenarians (box (A)) and proposed preferential pathways determining sphingolipids fate in aged subjects (B) and centenarians (C). Light blue boxes indicate lipids; white boxes indicate enzymes involved in the pathway, while unboxed acronyms indicate gene names. Gene names are highlighted in red when their mRNA expression in PBMC is higher in the AG vs. C comparison (box (B)) or in the C vs. AG comparison (box (C)). Aged subjects seem to be prone to increase ceramide levels through de novo biosynthesis (purple arrow), while centenarians convert sphingomyelin-derived ceramide into complex glyco/sphingolipids (yellow arrow).