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Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tg(fb)) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tg(fb) mice than in female Asm-tg(fb) mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tg(fb) mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression.
Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C₁₆ fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d₃ and L-serine-d₃. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B₁). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates – naturally occurring or chemically tailored – as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.
Dopamine (DA) and serotonin (SRT) are monoamine neurotransmitters that play a key role in regulating the central and peripheral nervous system. Their impaired metabolism has been implicated in several neurological disorders, such as Parkinson's disease and depression. Consequently, it is imperative to monitor changes in levels of these low-abundant neurotransmitters and their role in mediating disease. For the first time, a rapid, specific and sensitive isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of DA and SRT in the nematode Caenorhabditis elegans (C. elegans). This model organism offers a unique approach for studying the effect of various drugs and environmental conditions on neurotransmitter levels, given by the conserved DA and SRT biology, including synaptic release, trafficking and formation. We introduce a novel sample preparation protocol incorporating the usage of sodium thiosulfate in perchloric acid as extraction medium that assures high recovery of the relatively unstable neurotransmitters monitored. Moreover, the use of both deuterated internal standards and the multiple reaction monitoring (MRM) technique allows for unequivocal quantification. Thereby, to the best of our knowledge, we achieve a detection sensitivity that clearly exceeds those of published DA and SRT quantification methods in various matrices. We are the first to show that exposure of C elegans to the monoamine oxidase B (MAOB) inhibitor selegiline or the catechol-O-methyltransferase (COMT) inhibitor tolcapone, in order to block DA and SRT degradation, resulted in accumulation of the respective neurotransmitter. Assessment of a behavioral output of the dopaminergic system (basal slowing response) corroborated the analytical LC-MS/MS data. Thus, utilization of the C elegans model system in conjunction with our analytical method is well-suited to investigate drug-mediated modulation of the DA and SRT system in order to identify compounds with neuroprotective or regenerative properties. (C) 2015 Elsevier B.V. All rights reserved.
Chronic psychosocial stress adversely affects human morbidity and is a risk factor for inflammatory disorders, liver diseases, obesity, metabolic syndrome, and major depressive disorder (MDD). In recent studies, we found an association of MDD with an increase of acid sphingomyelinase (ASM) activity. Thus, we asked whether chronic psychosocial stress as a detrimental factor contributing to the emergence of MDD would also affect ASM activity and sphingolipid (SL) metabolism. To induce chronic psychosocial stress in male mice we employed the chronic subordinate colony housing (CSC) paradigm and compared them to non-stressed single housed control (SHC) mice. We determined Asm activity in liver and serum, hepatic SL concentrations as well as hepatic mRNA expression of genes involved in SL metabolism. We found that hepatic Asm activity was increased by 28% (P = 0.006) and secretory Asm activity by 47% (P = 0.002) in stressed mice. C16:0-Cer was increased by 40% (P = 0.008). Gene expression analysis further revealed an increased expression of tumor necrosis factor (TNF)-alpha (P = 0.009) and of several genes involved in SL metabolism (Cers5, P = 0.028; Cers6, P = 0.045; Gba, P = 0.049; Gba2, P = 0.030; Ormdl2, P = 0.034; Smpdl3B; P = 0.013). Our data thus provides first evidence that chronic psychosocial stress, at least in mice, induces alterations in SL metabolism, which in turn might be involved in mediating the adverse health effects of chronic psychosocial stress and peripheral changes occurring in mood disorders.
Background: Alcohol abuse is a major risk factor for somatic and neuropsychiatric diseases. Despite their potential clinical importance, little is known about the alterations of plasma glycerophospholipid (GPL) and sphingolipid (SPL) species associated with alcohol abuse.
Methods: Plasma GPL and SPL species were quantified using electrospray ionization tandem mass spectrometry in samples from 23 male alcohol-dependent patients before and after detoxification, as well as from 20 healthy male controls.
Results: A comparison of alcohol-dependent patients with controls revealed higher phosphatidylcholine (PC; P-value = 0.008) and phosphatidylinositol (PI; P-value = 0.001) concentrations in patients before detoxification, and higher PI (P-value = 0.001) and phosphatidylethanolamine (PE)-based plasmalogen (PEP; P-value = 0.003) concentrations after detoxification. Lysophosphatidylcholines (LPC) were increased by acute intoxication (P-value = 0.002). Sphingomyelin (SM) concentration increased during detoxification (P-value = 0.011). The concentration of SM 23:0 was lower in patients (P-value = 2.79 x 10(-5)), and the concentrations of ceramide Cer d18:1/16:0 and Cer d18:1/18:0 were higher in patients (P-value = 2.45 x 10(-5) and 3.73 x 10(-5)). Activity of lysosomal acid sphingomyelinase (ASM) in patients correlated positively with the concentrations of eight LPC species, while activity of secreted ASM was inversely correlated with several PE, PI and PC species, and positively correlated with the molar ratio of PC to SM (Pearson's r = 0.432; P-value = 0.039).
Conclusion: Plasma concentrations of numerous GPL and SPL species were altered in alcohol-dependent patients. These molecules might serve as potential biomarkers to improve the diagnosis of patients and to indicate health risks associated with alcohol abuse. Our study further indicates that there are strong interactions between plasma GPL concentrations and SPL metabolism. (C) 2015 Elsevier B.V. All rights reserved.
Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P.aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P.aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.
Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect.
Background & Aims: Exosomes are small membrane vesicles involved in intercellular communication. Hepatocytes are known to release exosomes, but little is known about their biological function. We sought to determine if exosomes derived from hepatocytes contribute to liver repair and regeneration after injury. Methods: Exosomes derived from primary murine hepatocytes were isolated and characterized biochemically and biophysically. Using cultures of primary hepatocytes, we tested whether hepatocyte exosomes induced proliferation of hepatocytes in vitro. Using models of ischemia/reperfusion injury and partial hepatectomy, we evaluated whether hepatocyte exosomes promote hepatocyte proliferation and liver regeneration in vivo. Results: Hepatocyte exosomes, but not exosomes from other liver cell types, induce dose-dependent hepatocyte proliferation in vitro and in vivo. Mechanistically, hepatocyte exosomes directly fuse with target hepatocytes and transfer neutral ceramidase and sphingosine kinase 2 (SK2) causing increased synthesis of sphingosine-1-phosphate (S1P) within target hepatocytes. Ablation of exosomal SK prevents the proliferative effect of exosomes. After ischemia/reperfusion injury, the number of circulating exosomes with proliferative effects increases. Conclusions: Our data shows that hepatocyte-derived exosomes deliver the synthetic machinery to form S1P in target hepatocytes resulting in cell proliferation and liver regeneration after ischemia/reperfusion injury or partial hepatectomy. These findings represent a potentially novel new contributing mechanism of liver regeneration and have important implications for new therapeutic approaches to acute and chronic liver disease. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes, can be irreversibly degraded by the membrane-bound S1P lyase (S1PL) yielding (2E)-hexadecenal and phosphoethanolamine. It is discussed that (2E)-hexadecenal is further oxidized to (2E)-hexadecenoic acid by the long-chain fatty aldehyde dehydrogenase ALDH3A2 (also known as FALDH) prior to activation via coupling to coenzyme A (CoA). Inhibition or defects in these enzymes, S1PL or FALDH, result in severe immunological disorders or the Sjogren-Larsson syndrome, respectively. Hence, it is of enormous importance to simultaneously determine the S1P breakdown product (2E)-hexadecenal and its fatty acid metabolites in biological samples. However, no method is available so far. Here, we present a sensitive and selective isotope-dilution high performance liquid chromatographyelectrospray ionizationquadrupole/time-of-flight mass spectrometry method for simultaneous quantification of (2E)-hexadecenal and its fatty acid metabolites following derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide. Optimized conditions for sample derivatization, chromatographic separation, and MS/MS detection are presented as well as an extensive method validation. Finally, our method was successfully applied to biological samples. We found that (2E)-hexadecenal is almost quantitatively oxidized to (2E)-hexadecenoic acid, that is further activated as verified by cotreatment of HepG2 cell lysates with (2E)-hexadecenal and the acyl-CoA synthetase inhibitor triacsin C. Moreover, incubations of cell lysates with deuterated (2E)-hexadecenal revealed that no hexadecanoic acid is formed from the aldehyde. Thus, our method provides new insights into the sphingolipid metabolism and will be useful to investigate diseases known for abnormalities in long-chain fatty acid metabolism, e.g., the Sjogren-Larsson syndrome, in more detail.
Within the last few decades, liquid chromatography-mass spectrometry (LC-MS) has become a preferred method for manifold issues in analytical biosciences, given its high selectivity and sensitivity. However, the analysis of fatty aldehydes, which are important components of cell metabolism, remains challenging. Usually, chemical derivatization prior to MS detection is required to enhance ionization efficiency. In this regard, the coupling of fatty aldehydes to hydrazines like 2,4-dinitrophenylhydrazine (DNPH) is a common approach. Additionally, hydrazones readily react with fatty aldehydes to form stable derivatives, which can be easily separated using high-performance liquid chromatography (HPLC) and subsequently detected by MS. Here, we exemplarily present the quantification of the long-chain fatty aldehyde (2E)-hexadecenal, a break-down product of the bioactive lipid sphingosine 1-phosphate (S1P), after derivatization with 2-diphenylacetyl-1,3-indandione-1-hydrazone (DAIH) via isotope-dilution HPLC-electrospray ionization-quadrupole/time-of-flight (ESI-QTOF) MS. Moreover, we show that the addition of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC hydrochloride) as a coupling agent allows for simultaneous determination of fatty aldehydes and fatty acids as DAIH derivatives. Taking advantage of this, we describe in detail how to monitor the degradation of (2E)-hexadecenal and the concurrent formation of its oxidation product (2E)-hexadecenoic acid in lysates of human hepatoblastoma (HepG2) cells within this chapter.