Refine
Has Fulltext
- yes (17)
Document Type
- Postprint (17) (remove)
Language
- English (17)
Is part of the Bibliography
- yes (17) (remove)
Keywords
- acid sphingomyelinase (3)
- sphingolipids (3)
- acid ceramidase (2)
- ceramide (2)
- 1-aminodecylidene bis-phosphonic acid (1)
- Drug delivery (1)
- Epigenetics (1)
- Farber disease (1)
- Gestational diabetes (1)
- Global DNA methylation (1)
Institute
- Institut für Ernährungswissenschaft (17) (remove)
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.
Loss of pdr-1/parkin influences Mn homeostasis through altered ferroportin expression in C. elegans
(2015)
Overexposure to the essential metal manganese (Mn) can result in an irreversible condition known as manganism that shares similar pathophysiology with Parkinson's disease (PD), including dopaminergic (DAergic) cell loss that leads to motor and cognitive impairments. However, the mechanisms behind this neurotoxicity and its relationship with PD remain unclear. Many genes confer risk for autosomal recessive, early-onset PD, including the parkin/PARK2 gene that encodes for the E3 ubiquitin ligase Parkin. Using Caenorhabditis elegans (C. elegans) as an invertebrate model that conserves the DAergic system, we previously reported significantly increased Mn accumulation in pdr-1/parkin mutants compared to wildtype (WT) animals. For the current study, we hypothesize that this enhanced accumulation is due to alterations in Mn transport in the pdr-1 mutants. While no change in mRNA expression of the major Mn importer proteins (smf-1-3) was found in pdr-1 mutants, significant downregulation in mRNA levels of the putative Mn exporter ferroportin (fpn-1.1) was observed. Using a strain overexpressing fpn-1.1 in worms lacking pdr-1, we show evidence for attenuation of several endpoints of Mn-induced toxicity, including survival, metal accumulation, mitochondrial copy number and DAergic integrity, compared to pdr-1 mutants alone. These changes suggest a novel role of pdr-1 in modulating Mn export through altered transporter expression, and provides further support of metal dyshomeostasis as a component of Parkinsonism pathophysiology.
Background: Gestational diabetes mellitus (GDM) is associated with adverse pregnancy outcomes. It is known that GDM is associated with an altered placental function and changes in placental gene regulation. More recent studies demonstrated an involvement of epigenetic mechanisms. So far, the focus regarding placental epigenetic changes in GDM was set on gene-specific DNA methylation analyses. Studies that robustly investigated placental global DNA methylation are lacking. However, several studies showed that tissue-specific alterations in global DNA methylation are independently associated with type 2 diabetes. Thus, the aim of this study was to characterize global placental DNA methylation by robustly measuring placental DNA 5-methylcytosine (5mC) content and to examine whether differences in placental global DNA methylation are associated with GDM.
Methods: Global DNA methylation was quantified by the current gold standard method, LC-MS/MS. In total, 1030 placental samples were analyzed in this single-center birth cohort study.
Results: Mothers with GDM displayed a significantly increased global placental DNA methylation (3.22 ± 0.63 vs. 3.00 ± 0.46 %; p = 0.013; ±SD). Bivariate logistic regression showed a highly significant positive correlation between global placental DNA methylation and the presence of GDM (p = 0.0009). Quintile stratification according to placental DNA 5mC levels revealed that the frequency of GDM was evenly distributed in quintiles 1–4 (2.9–5.3 %), whereas the frequency in the fifth quintile was significantly higher (10.7 %; p = 0.003). Bivariate logistic models adjusted for maternal age, BMI, ethnicity, recurrent miscarriages, and familiar diabetes predisposition clearly demonstrated an independent association between global placental DNA hypermethylation and GDM. Furthermore, an ANCOVA model considering known predictors of DNA methylation substantiated an independent association between GDM and placental DNA methylation.
Conclusions: This is the first study that employed a robust quantitative assessment of placental global DNA methylation in over a thousand placental samples. The study provides large scale evidence that placental global DNA hypermethylation is associated with GDM, independent of established risk factors.
Two decades ago, sphingosine 1-phosphate (S1P) was discovered as a novel bioactive molecule that regulates a variety of cellular functions. The plethora of S1P-mediated effects is due to the fact that the sphingolipid not only modulates intracellular functions but also acts as a ligand of G protein-coupled receptors after secretion into the extracellular environment. In the plasma, S1P is found in high concentrations, modulating immune cell trafficking and vascular endothelial integrity. The liver is engaged in modulating the plasma S1P content, as it produces apolipoprotein M, which is a chaperone for the S1P transport. Moreover, the liver plays a substantial role in glucose and lipid homeostasis. A dysfunction of glucose and lipid metabolism is connected with the development of liver diseases such as hepatic insulin resistance, non-alcoholic fatty liver disease, or liver fibrosis. Recent studies indicate that S1P is involved in liver pathophysiology and contributes to the development of liver diseases. In this review, the current state of knowledge about S1P and its signaling in the liver is summarized with a specific focus on the dysregulation of S1P signaling in obesity-mediated liver diseases. Thus, the modulation of S1P signaling can be considered as a potential therapeutic target for the treatment of hepatic diseases.
Dermal Delivery of the High-Molecular-Weight Drug Tacrolimus by Means of Polyglycerol-Based Nanogels
(2019)
Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions.
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.
Inhibition of acid sphingomyelinase (ASM), a lysosomal enzyme that catalyzes the hydrolysis of sphingomyelin into ceramide and phosphorylcholine, may serve as an investigational tool or a therapeutic intervention to control many diseases. Specific ASM inhibitors are currently not sufficiently characterized. Here, we found that 1-aminodecylidene bis-phosphonic acid (ARC39) specifically and efficiently (>90%) inhibits both lysosomal and secretory ASM in vitro. Results from investigating sphingomyelin phosphodiesterase 1 (SMPD1/Smpd1) mRNA and ASM protein levels suggested that ARC39 directly inhibits ASM's catalytic activity in cultured cells, a mechanism that differs from that of functional inhibitors of ASM. We further provide evidence that ARC39 dose- and time-dependently inhibits lysosomal ASM in intact cells, and we show that ARC39 also reduces platelet- and ASM-promoted adhesion of tumor cells. The observed toxicity of ARC39 is low at concentrations relevant for ASM inhibition in vitro, and it does not strongly alter the lysosomal compartment or induce phospholipidosis in vitro. When applied intraperitoneally in vivo, even subtoxic high doses administered short-term induced sphingomyelin accumulation only locally in the peritoneal lavage without significant accumulation in plasma, liver, spleen, or brain. These findings require further investigation with other possible chemical modifications. In conclusion, our results indicate that ARC39 potently and selectively inhibits ASM in vitro and highlight the need for developing compounds that can reach tissue concentrations sufficient for ASM inhibition in vivo.