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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.
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.
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.
Due to the low cutaneous bioavailability of tacrolimus (TAC), penetration enhancers are used to improve its penetration into the skin. However, poor loading capacity, non-biodegradability, toxicity, and in some cases inefficient skin penetration are challenging issues that hamper their applications for the dermal TAC delivery. Here we present poly(lactide-co-glycerol) (PLG) as a water soluble, biodegradable, and biocompatible TAC-carrier with high loading capacity (14.5% w/w for TAC) and high drug delivery efficiencies into the skin. PLG was synthesized by cationic ring-opening copolymerization of a mixture of glycidol and lactide and showed 35 nm and 300 nm average sizes in aqueous solutions before and after loading of TAC, respectively. Delivery experiments on human skin, quantified by fluorescence microscopy and LC-MS/MS, showed a high ability for PLG to deposit Nile red and TAC into the stratum corneum and viable epidermis of skin in comparison with Protopic (R) (0.03% w/w, TAC ointment). The cutaneous distribution profile of delivered TAC proved that 80%, 16%, and 4% of the cutaneous drug level was deposited in the stratum corneum, viable epidermis, and upper dermis, respectively. TAC delivered by PLG was able to efficiently decrease the IL-2 and TSLP expressions in human skin models. Taking advantage of the excellent physicochemical and biological properties of PLG, it can be used for efficient dermal TAC delivery and potential treatment of inflammatory skin diseases.
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.
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.
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.
We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N-2-(1-MIM)-dG, N-6-(1-MIM)-dA] in six tissues, as well as protein adducts [tau N-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N-2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N-6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.
Pak Choi Fed to Mice: Formation of DNA Adducts and Influence on Xenobiotic-Metabolizing Enzymes
(2015)
Ventilator-induced lung injury is aggravated by antibiotic mediated microbiota depletion in mice
(2018)
BackgroundAntibiotic exposure alters the microbiota, which can impact the inflammatory immune responses. Critically ill patients frequently receive antibiotic treatment and are often subjected to mechanical ventilation, which may induce local and systemic inflammatory responses and development of ventilator-induced lung injury (VILI). The aim of this study was to investigate whether disruption of the microbiota by antibiotic therapy prior to mechanical ventilation affects pulmonary inflammatory responses and thereby the development of VILI.MethodsMice underwent 6-8weeks of enteral antibiotic combination treatment until absence of cultivable bacteria in fecal samples was confirmed. Control mice were housed equally throughout this period. VILI was induced 3 days after completing the antibiotic treatment protocol, by high tidal volume (HTV) ventilation (34ml/kg; positive end-expiratory pressure=2 cmH(2)O) for 4h. Differences in lung function, oxygenation index, pulmonary vascular leakage, macroscopic assessment of lung injury, and leukocyte and lymphocyte differentiation were assessed. Control groups of mice ventilated with low tidal volume and non-ventilated mice were analyzed accordingly.ResultsAntibiotic-induced microbiota depletion prior to HTV ventilation led to aggravation of VILI, as shown by increased pulmonary permeability, increased oxygenation index, decreased pulmonary compliance, enhanced macroscopic lung injury, and increased cytokine/chemokine levels in lung homogenates.ConclusionsDepletion of the microbiota by broad-spectrum antibiotics prior to HTV ventilation renders mice more susceptible to developing VILI, which could be clinically relevant for critically ill patients frequently receiving broad-spectrum antibiotics.