Study of sphingolipid-mediated signaling and metabolism during aging

Sphingolipids are a class of bioactive signaling molecules that regulate key cellular processes including cell growth, senescence and apoptosis, and have been implicated in age-related neurodegeneration. Previous studies have reported elevated ceramide levels in the brain of old rodents, but a systematic investigation of the impact of age on brain sphingolipid metabolism was still lacking. In the present study we quantified 17 key sphingolipid species in the hippocampus of young (3 months), middle-aged (12 months) and old (21 months) male and female mice. Lipids were extracted and quantified by liquid chromatography/mass spectrometry; transcription of enzymes involved in sphingolipid biosynthesis was evaluated by qPCR. Age-dependent changes of multiple sphingolipid species - including ceramide (d18:1/18:0), sphingomyelin (d34:1), hexosylceramide (d18:1/16:0), ceramide (d18:1/24:0) - were found in mice of both sexes. Moreover, sex-dependent changes were seen with hexosylceramide (d18:1/18:0), ceramide (d18:1/22:0), sphingomyelin (d36:1) and sphingomyelin (d42:1). Importantly, an age-dependent accumulation of sphingolipids containing nervonic acid (24:1) was observed in 21 month-old male (p = 0.04) and female mice (p < 0.001). Consistent with this increase, transcription of the nervonic acid-synthesizing enzyme, stearoyl-CoA desaturase (Scd1 and Scd2), was upregulated in 21 month-old female mice (Scd1 p = 0.006; Scd2 p = 0.009); a similar trend was observed in males (Scd1 p = 0.07). In conclusion, the results suggest that aging is associated with profound sex-dependent and -independent changes in hippocampal sphingolipid profile. The results also highlight the need to examine the contribution of sphingolipids, and particularly of those containing nervonic acid, in normal and pathological brain aging. Nevertheless, also the circulating ceramids are altered in persons affected by age-dependent pathologies such as metabolic syndrome, mild cognitive impairment and Alzheimer\u2019s disease, but the potential impact of age and gender on plasma ceramide trajectories in healthy subjects has not been systematically examined. In this study we quantified a panel of circulating ceramides and dihydroceramides in a cohort of 164 healthy subjects (84 female, 80 male; 19-80 years of age). The results show that plasma ceramide levels are significantly lower (p < 0.05) in pre-menopausal women (aged 20-54 years) compared to age-matched men (aged 19-54 years). This difference disappears after menopause, such that plasma ceramide levels in post-menopausal women (aged 47-78 years) are statistically identical to those measured in aged-matched men (aged 55-80 years). In women of all ages, but not in men, circulating levels of ceramide (d18:1/24:1) were negatively correlated with plasma estradiol levels. Finally, in vitro experiment showed that incubation with estradiol (10 nM, 24 h) lowers ceramide levels in the human MCF7 breast cancer cell line. Together the results suggest the existence of gender- and age-dependent alterations in circulating ceramide concentrations, which are dependent on estradiol. In addition to my previous study on rodent model, the present work introduces menopause and fluctuating estradiol levels as new variables to keep into account in the study of aging. Aging is the main risk factor for the development of neurodegenerative diseases such as Alzheimer's disease but also chronic diseases such as metabolic syndrome. Indeed, it is becoming increasingly evident that cellular and biochemical alterations observed in metabolic syndrome like, among others, alterations in lipid mediators, may represent a pathological bridge between age-related neurological disorders and metabolic syndrome. Sphingolipids have been implicated in the pathogenesis of metabolic dysfunction, but physiological signals regulating their formation and deactivations in hypothalamus are unknown. Hypothalamus is an especially important node in central and peripheral regulation of feeding behavior. We studied the effect of high-fat diet (HFD) or food deprivation (FD) on sphingolipid levels and on the expression of enzymes involved in sphingolipid metabolism in the hypothalamus. To study the effect of HFD, mice were divided into two groups: standard diet (2.66 kcal/g) and HFD (5.24 kcal/g) and killed at different time points (1-3-7-14-28 days). To study the effect of fasting, male mice were subjected to 4 feeding conditions: 1) free feeding (FF); 2) 12h food deprivation (FD); 3) 1h refeeding after FD; 4) 6h refeeding after FD. Hypothalamic sphingolipids were extracted and quantified by LC-MS/MS. Transcription of enzymes involved in sphingolipid biosynthesis was evaluated by qPCR. After 1 day and 14 days, mice exposed to a HFD showed lower levels of ceramide (d18:1/24:0), its precursor dihydroceramide (d18:0/24:0), and ceramide (d18:1/24:1) compared to mice fed standard diet. Significant decrease in sphingosine-1-phosphate (SO-1-P) was also observed after 7 and 14 days of HFD. Relative to FF, fasting: 1) decreased SO-1-P levels; 2) increased sphingosine, the precursor of sphingosine-1-phosphate; 3) reduced the levels of dihydroceramide (d18:0/18:0), a product of de novo ceramide biosynthesis; 4) down-regulated transcription of sphingosine kinases (SphK) and ceramide synthase 1 (CerS1). Our results suggest that hypothalamic levels of SO-1-P, its precursor sphingosine and enzymes involved in their metabolism (SphK) are influenced by feeding status. Feeding also regulates the de novo synthesis of sphingolipid, suggesting additional roles for these lipids in the control of energy balance. Finally, a single intracerebroventricular injection of an acid ceramidase inhibitor, ARN14974, which has been shown to imbalance the ceramide/sphingosine-1-phosphate rheostat, was able to alter some parameters of feeding behavior such as meal size. The aim of my studies was to explore the complexity of sphingolipid metabolism and understand the role of sphingolipids as lipid-derived mediators of cell signaling in physiological or altered conditions in rodent model and in human. Aging and feeding states are strongly correlated since aging has been associated with development of obesity and metabolic disorders, which depend, in turn, on altered feeding status; on the other hand, hyperphagia or fasting may have respectively detrimental or beneficial effect on aging and longevity. Furthermore, since a variety of studies have been published on the relevance of dimorphism in the development of age-related disorders, I pursued my research keeping into account sex-related differences. Brain areas and plasma have been chosen as target tissues for my studies. My research has been focused on specific cerebral areas involved respectively in aging and cognition (hippocampus) and feeding control and energy balance (hypothalamus). To investigate the role of ceramides in human, I had access to plasma from healthy subjects recruited by Santa Lucia Foundation. Plasma is widely used to assess biomarkers of pathological states in human studies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS), a unique technology with the requisite of specificity, sensitivity and quantitative precision capabilities, allowed me to qualify and quantify target sphingolipids

Feeding Stimulates Sphingosine-1-Phosphate Mobilization in Mouse Hypothalamus.

Previous studies have shown that the sphingolipid-derived mediator sphingosine-1-phosphate (S1P) reduces food intake by activating G protein-coupled S1P receptor-1 (S1PR1) in the hypothalamus. Here, we examined whether feeding regulates hypothalamic mobilization of S1P and other sphingolipid-derived messengers. We prepared lipid extracts from the hypothalamus of C57Bl6/J male mice subjected to one of four conditions: free feeding, 12 h fasting, and 1 h or 6 h refeeding. Liquid chromatography/tandem mass spectrometry was used to quantify various sphingolipid species, including sphinganine (SA), sphingosine (SO), and their bioactive derivatives SA-1-phosphate (SA1P) and S1P. In parallel experiments, transcription of S1PR1 (encoded in mice by the S1pr1 gene) and of key genes of sphingolipid metabolism (Sptlc2, Lass1, Sphk1, Sphk2) was measured by RT-PCR. Feeding increased levels of S1P (in pmol-mg-1 of wet tissue) and SA1P. This response was accompanied by parallel changes in SA and dihydroceramide (d18:0/18:0), and was partially (SA1P) or completely (S1P) reversed by fasting. No such effects were observed with other sphingolipid species targeted by our analysis. Feeding also increased transcription of Sptlc2, Lass1, Sphk2, and S1pr1. Feeding stimulates mobilization of endogenous S1PR1 agonists S1P and SA1P in mouse hypothalamus, via a mechanism that involves transcriptional up-regulation of de novo sphingolipid biosynthesis. The results support a role for sphingolipid-mediated signaling in the central control of energy balance

Elevated plasma ceramide levels in post-menopausal women: a cross-sectional study.

Circulating ceramide levels are abnormally elevated in age-dependent pathologies such as cardiovascular diseases, obesity and Alzheimer's disease. Nevertheless, the potential impact of age on plasma ceramide levels has not yet been systematically examined. In the present study, we quantified a focused panel of plasma ceramides and dihydroceramides in a cohort of 164 subjects (84 women) 19 to 80 years of age. After adjusting for potential confounders, multivariable linear regression analysis revealed a positive association between age and ceramide (d18:1/24:0) (β (SE) = 5.67 (2.38); p = .0198) and ceramide (d18:1/24:1) (β (SE) = 2.88 (.61); p &lt; .0001) in women, and between age and ceramide (d18:1/24:1) in men (β (SE) = 1.86 (.77); p = .0179). In women of all ages, but not men, plasma ceramide (d18:1/24:1) was negatively correlated with plasma estradiol (r = -0.294; p = .007). Finally, in vitro experiments in human cancer cells expressing estrogen receptors showed that incubation with estradiol (10 nM, 24 h) significantly decreased ceramide accumulation. Together, the results suggest that aging is associated with an increase in circulating ceramide levels, which in post-menopausal women is at least partially associated with lower estradiol levels

Fast and Sensitive Quantification of Δ9-Tetrahydrocannabinol and Its Main Oxidative Metabolites by Liquid Chromatography/Tandem Mass Spectrometry

Introduction: Few animal studies have evaluated the pharmacological effects of Δ9-tetrahydrocannabinol (THC) in relation to its pharmacokinetic properties. Understanding this relationship is essential, however, if comparisons are to be drawn across conditions-such as sex, age, and route of administration-which are associated with variations in the absorption, metabolism, and distribution of THC. As a first step toward addressing this gap, in this report, we describe a rapid, sensitive, and accurate method for the quantification of THC and its main oxidative metabolites, and apply it to representative rodent tissues. Materials and Methods: The sample workup procedure consisted of two steps: bulk protein precipitation with cold acetonitrile (ACN) followed by phospholipid removal by elution through Captiva-Enhanced Matrix Removal cartridges. The liquid chromatography/tandem mass spectrometry (LC/MS-MS) protocol utilized a commercially available C18 reversed-phase column and a simple methanol/water gradient system. The new method was validated following Food and Drug Administration (FDA) guidelines, and was applied to the quantification of THC and its main oxidative metabolites-11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (11-COOH-THC)-in plasma and brain of mice treated with a single intraperitoneal dose of THC (10 mg/kg). Results: ACN precipitation and column elution effectively depleted matrix constituents-most notably choline-containing phospholipids-which are known to interfere with THC analysis, with average recovery values of &gt;85% for plasma and &gt;80% for brain. The LC conditions yielded baseline separation of all analytes in a total run time of 7 min (including re-equilibration). The 10-point calibration curves showed excellent linearity (R 2&gt;0.99) over a wide range of concentrations (1-1000 pmol/100 μL). Lowest limit of quantification was 2 pmol/100 μL for all analytes, and lowest limits of detection were 0.5 pmol/100 μL for THC and 11-OH-THC, and 1 pmol/100 μL for 11-COOH-THC. Intraday and interday accuracy and precision values were within the FDA-recommended range (±15% of nominal concentration). An application of the method to adult male mice is presented. Conclusions: We present a fast and sensitive method for the analysis of THC, which should facilitate studies aimed at linking the pharmacokinetics and pharmacodynamics of this compound in animal models

Feeding Stimulates Sphingosine-1-Phosphate Mobilization in Mouse Hypothalamus.

Previous studies have shown that the sphingolipid-derived mediator sphingosine-1-phosphate (S1P) reduces food intake by activating G protein-coupled S1P receptor-1 (S1PR1) in the hypothalamus. Here, we examined whether feeding regulates hypothalamic mobilization of S1P and other sphingolipid-derived messengers. We prepared lipid extracts from the hypothalamus of C57Bl6/J male mice subjected to one of four conditions: free feeding, 12 h fasting, and 1 h or 6 h refeeding. Liquid chromatography/tandem mass spectrometry was used to quantify various sphingolipid species, including sphinganine (SA), sphingosine (SO), and their bioactive derivatives SA-1-phosphate (SA1P) and S1P. In parallel experiments, transcription of S1PR1 (encoded in mice by the S1pr1 gene) and of key genes of sphingolipid metabolism (Sptlc2, Lass1, Sphk1, Sphk2) was measured by RT-PCR. Feeding increased levels of S1P (in pmol-mg-1 of wet tissue) and SA1P. This response was accompanied by parallel changes in SA and dihydroceramide (d18:0/18:0), and was partially (SA1P) or completely (S1P) reversed by fasting. No such effects were observed with other sphingolipid species targeted by our analysis. Feeding also increased transcription of Sptlc2, Lass1, Sphk2, and S1pr1. Feeding stimulates mobilization of endogenous S1PR1 agonists S1P and SA1P in mouse hypothalamus, via a mechanism that involves transcriptional up-regulation of de novo sphingolipid biosynthesis. The results support a role for sphingolipid-mediated signaling in the central control of energy balance

Feeding Stimulates Sphingosine-1-Phosphate Mobilization in Mouse Hypothalamus

Previous studies have shown that the sphingolipid-derived mediator sphingosine-1-phosphate (S1P) reduces food intake by activating G protein-coupled S1P receptor-1 (S1PR1) in the hypothalamus. Here, we examined whether feeding regulates hypothalamic mobilization of S1P and other sphingolipid-derived messengers. We prepared lipid extracts from the hypothalamus of C57Bl6/J male mice subjected to one of four conditions: free feeding, 12 h fasting, and 1 h or 6 h refeeding. Liquid chromatography/tandem mass spectrometry was used to quantify various sphingolipid species, including sphinganine (SA), sphingosine (SO), and their bioactive derivatives SA-1-phosphate (SA1P) and S1P. In parallel experiments, transcription of S1PR1 (encoded in mice by the S1pr1 gene) and of key genes of sphingolipid metabolism (Sptlc2, Lass1, Sphk1, Sphk2) was measured by RT-PCR. Feeding increased levels of S1P (in pmol-mg&minus;1 of wet tissue) and SA1P. This response was accompanied by parallel changes in SA and dihydroceramide (d18:0/18:0), and was partially (SA1P) or completely (S1P) reversed by fasting. No such effects were observed with other sphingolipid species targeted by our analysis. Feeding also increased transcription of Sptlc2, Lass1, Sphk2, and S1pr1. Feeding stimulates mobilization of endogenous S1PR1 agonists S1P and SA1P in mouse hypothalamus, via a mechanism that involves transcriptional up-regulation of de novo sphingolipid biosynthesis. The results support a role for sphingolipid-mediated signaling in the central control of energy balance