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Institute
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.
Background/Aims:
Obesity is a main risk factor for the development of hepatic insulin resistance and it is accompanied by adipocyte hypertrophy and an elevated expression of different adipokines such as autotaxin (ATX). ATX converts lysophosphatidylcholine to lysophosphatidic acid (LPA) and acts as the main producer of extracellular LPA. This bioactive lipid regulates a broad range of physiological and pathological responses by activation of LPA receptors (LPA1-6).
Methods:
The activation of phosphatidylinositide 3-kinases (PI3K) signaling (Akt and GSK-3ß) was analyzed via western blotting in primary rat hepatocytes. Incorporation of glucose into glycogen was measured by using radio labeled glucose. Real-time PCR analysis and pharmacological modulation of LPA receptors were performed. Human plasma LPA levels of obese (BMI > 30, n = 18) and normal weight individuals (BMI 18.5-25, n = 14) were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS).
Results:
Pretreatment of primary hepatocytes with LPA resulted in an inhibition of insulin-mediated Gck expression, PI3K activation and glycogen synthesis. Pharmacological approaches revealed that the LPA3-receptor subtype is responsible for the inhibitory effect of LPA on insulin signaling. Moreover, human plasma LPA concentrations (16: 0 LPA) of obese participants (BMI > 30) are significantly elevated in comparison to normal weight individuals (BMI 18.5-25).
Conclusion:
LPA is able to interrupt insulin signaling in primary rat hepatocytes via the LPA3 receptor subtype. Moreover, the bioactive lipid LPA (16: 0) is increased in obesity.
Insulin is the main anabolic hormone secreted by 13-cells of the pancreas stimulating the assimilation and storage of glucose in muscle and fat cells. It modulates the postprandial balance of carbohydrates, lipids and proteins via enhancing lipogenesis, glycogen and protein synthesis and suppressing glucose generation and its release from the liver. Resistance to insulin is a severe metabolic disorder related to a diminished response of peripheral tissues to the insulin action and signaling. This leads to a disturbed glucose homeostasis that precedes the onset of type 2 diabetes (T2D), a disease reaching epidemic proportions. A large number of studies reported an association between elevated circulating fatty acids and the development of insulin resistance. The increased fatty acid lipid flux results in the accumulation of lipid droplets in a variety of tissues. However, lipid intermediates such as diacylglycerols and ceramides are also formed in response to elevated fatty acid levels. These bioactive lipids have been associated with the pathogenesis of insulin resistance. More recently, sphingosine 1-phosphate (S1P), another bioactive sphingolipid derivative, has also been shown to increase in T2D and obesity. Although many studies propose a protective role of S1P metabolism on insulin signaling in peripheral tissues, other studies suggest a causal role of S1P on insulin resistance. In this review, we critically summarize the current state of knowledge of S1P metabolism and its modulating role on insulin resistance. A particular emphasis is placed on S1P and insulin signaling in hepatocytes, skeletal muscle cells, adipocytes and pancreatic 13-cells. In particular, modulation of receptors and enzymes that regulate S1P metabolism can be considered as a new therapeutic option for the treatment of insulin resistance and T2D.
Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C-elegans
(2015)
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.
Farber disease is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments for Farber disease are clinically available, and affected patients have a severely shortened lifespan. We have recently reported a novel acid ceramidase deficiency model that mirrors the human disease closely. Acid sphingomyelinase is the enzyme that generates ceramide upstream of acid ceramidase in the lysosomes. Using our acid ceramidase deficiency model, we tested if acid sphingomyelinase could be a potential novel therapeutic target for the treatment of Farber disease. A number of functional acid sphingomyelinase inhibitors are clinically available and have been used for decades to treat major depression. Using these as a therapeutic for Farber disease, thus, has the potential to improve central nervous symptoms of the disease as well, something all other treatment options for Farber disease can’t achieve so far. As a proof-of-concept study, we first cross-bred acid ceramidase deficient mice with acid sphingomyelinase deficient mice in order to prevent ceramide accumulation. Double-deficient mice had reduced ceramide accumulation, fewer disease manifestations, and prolonged survival. We next targeted acid sphingomyelinase pharmacologically, to test if these findings would translate to a setting with clinical applicability. Surprisingly, the treatment of acid ceramidase deficient mice with the acid sphingomyelinase inhibitor amitriptyline was toxic to acid ceramidase deficient mice and killed them within a few days of treatment. In conclusion, our study provides the first proof-of-concept that acid sphingomyelinase could be a potential new therapeutic target for Farber disease to reduce disease manifestations and prolong survival. However, we also identified previously unknown toxicity of the functional acid sphingomyelinase inhibitor amitriptyline in the context of Farber disease, strongly cautioning against the use of this substance class for Farber disease patients.
Recent investigations propose the acid sphingomyelinase (ASM)/ceramide system as a novel target for antidepressant action. ASM catalyzes the breakdown of the abundant membrane lipid sphingomyelin to the lipid messenger ceramide. This ASM‐induced lipid modification induces a local shift in membrane properties, which influences receptor clustering and downstream signaling. Canonical transient receptor potential channels 6 (TRPC6) are non‐selective cation channels located in the cell membrane that play an important role in dendritic growth, synaptic plasticity and cognition in the brain. They can be activated by hyperforin, an ingredient of the herbal remedy St. John’s wort for treatment of depression disorders. Because of their role in the context of major depression, we investigated the crosstalk between the ASM/ceramide system and TRPC6 ion channels in a pheochromocytoma cell line 12 neuronal cell model (PC12 rat pheochromocytoma cell line). Ca2+ imaging experiments indicated that hyperforin‐induced Ca2+ influx through TRPC6 channels is modulated by ASM activity. While antidepressants, known as functional inhibitors of ASM activity, reduced TRPC6‐mediated Ca2+ influx, extracellular application of bacterial sphingomyelinase rebalanced TRPC6 activity in a concentration‐related way. This effect was confirmed in whole‐cell patch clamp electrophysiology recordings. Lipidomic analyses revealed a decrease in very long chain ceramide/sphingomyelin molar ratio after ASM inhibition, which was connected with changes in the abundance of TRPC6 channels in flotillin‐1–positive lipid rafts as visualized by western blotting. Our data provide evidence that the ASM/ceramide system regulates TRPC6 channels likely by controlling their recruitment to specific lipid subdomains and thereby fine‐tuning their physical properties.
Background: The pathophysiology behind the subacute but persistent hypercoagulable state after traumatic brain injury (TBI) is poorly understood but contributes to morbidity induced by venous thromboembolism. Because platelets and their microvesicles have been hypothesized to play a role in post-traumatic hypercoagulability, administration of commonly used agents may ameliorate this coagulability. We hypothesized that utilization of aspirin, ketorolac, amitriptyline, unfractionated heparin, or enoxaparin would modulate the platelet aggregation response after TBI. Methods: Concussive TBI was induced by weight drop. Mice were then randomized to receive aspirin, ketorolac, amitriptyline, heparin, enoxaparin, or saline control at 2 and 8 h after TBI. Mice were sacrificed at 6 or 24 h after injury to determine coagulability by rotational thromboelastometry (ROTEM), platelet function testing with impedance aggregometry, and microvesicle enumeration. Platelet sphingolipid metabolites were analyzed by mass spectrometry. Results: ROTEM demonstrated increased platelet contribution to maximum clot firmness at 6 h after TBI in mice that received aspirin or amitriptyline, but this did not persist at 24 h. By contrast, adenosine diphosphate- and arachidonic acid-induced platelet aggregation at 6 h was significantly lower in mice receiving ketorolac, aspirin, and amitriptyline compared with mice receiving saline at 6 h after injury and only arachidonic acid-initiated platelet aggregation was decreased by aspirin at 24 h. There were no differences in microvesicle production at either time point. Platelet sphingosine-1-phosphate levels were decreased at 6 h in the group receiving amitriptyline and increased at 24 h along with platelet ceramide levels at 24 h in the amitriptyline group. Conclusion: After TBI, amitriptyline decreased platelet aggregability and increased contribution to clot in a manner similar to aspirin. The amitriptyline effects on platelet function and sphingolipid metabolites may represent a possible role of the acid sphingomyelinase in the hypercoagulability observed after injury. In addition, inhibition of platelet reactivity may be an underappreciated benefit of low molecular weight heparins, such as enoxaparin. (C) 2019 Elsevier Inc. All rights reserved.
Selenium increases hepatic DNA methylation and modulates one-carbon metabolism in the liver of mice
(2017)
The average intake of the essential trace element selenium (Se) is below the recommendation in most European countries, possibly causing sub-optimal expression of selenoproteins. It is still unclear how a suboptimal Se status may affect health. To mimic this situation, mice were fed one of three physiologically relevant amounts of Se. We focused on the liver, the organ most sensitive to changes in the Se supply indicated by hepatic glutathione peroxidase activity. In addition, liver is the main organ for synthesis of methyl groups and glutathione via one-carbon metabolism. Accordingly, the impact of Se on global DNA methylation, methylation capacity, and gene expression was assessed. We observed higher global DNA methylation indicated by LINE1 methylation, and an increase of the methylation potential as indicated by higher S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio and by elevated mRNA expression of serine hydroxymethyltransferase in both or either of the Se groups. Furthermore, increasing the Se supply resulted in higher plasma concentrations of triglycerides. Hepatic expression of glycolytic and lipogenic genes revealed consistent Se dependent up-regulation of glucokinase. The sterol regulatory element-binding transcription factor 1 (Srebf1) was also up-regulated by Se. Both effects were confirmed in primary hepatocytes. In contrast to the overall Se-dependent increase of methylation capacity, the up-regulation of Srebf1 expression was paralleled by reduced local methylation of a specific CpG site within the Srebf1 gene. Thus, we provided evidence that Se-dependent effects on lipogenesis involve epigenetic mechanisms. (C) 2017 The Authors. Published by Elsevier Inc.
As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90%) in PBL and 70-80% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5-6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus.