No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Increased Acid Sphingomyelinase Activity in Peripheral Blood Cells of Acutely Intoxicated Patients With Alcohol Dependence
Abstract
Acid sphingomyelinase (ASM; EC 3.1.4.12) hydrolyses membrane sphingomyelin into the bioactive lipid ceramide and is thus involved in different cellular processes such as differentiation, immunity, or cell death. Activation of ASM has been reported in particular in conjunction with the cellular stress response to several external stimuli, and increased ASM activity was observed in a variety of human diseases. Ethanol-induced activation of ASM has been observed in different cell culture systems, thus raising the question about the effect of alcohol intoxication in human subjects on ASM activity in vivo.
We determined ASM activity in peripheral blood mononucleated cells of 27 patients suffering from alcohol dependence. Patients were classified according to their blood alcohol concentration at admission, and ASM activity was determined repeatedly from all patients during alcohol withdrawal.
Acutely intoxicated patients displayed significantly higher ASM activity than patients in early abstinence (Mann-Whitney U test: Z = - 2.6, p = 0.009). ASM activity declined in acutely intoxicated patients to normal values with the transition from the intoxicated state to early abstinence (Wilcoxon test: Z = -2.7, p = 0.007). At the end of withdrawal, ASM activity was significantly increased again compared to the early phase of abstinence in both patient groups (Wilcoxon test: Z = -2.691, p = 0.007 and Z = -2.275, p = 0.023, respectively).
Alcohol-induced activation of ASM occurs in human subjects and might be responsible for deleterious effects of ethanol intoxication. Chronic alcohol abuse may induce deregulation of sphingomyelin metabolism in general, and this impairment may cause side effects during withdrawal from alcohol.
... Daraus lässt sich schließen, dass Alkohol die A-SMase zu aktivieren scheint. Die daraus resultierende erhöhte Ceramidkonzentration könnte bei der Entstehung einer alkoholinduzierten Major-Depression eine Rolle spielen (Reichel et al., 2011(Reichel et al., , 2010. Ceramide könnten außerdem als Schlüsselmechanismus für die alkoholinduzierte Apoptose in Zellen der Leber, des Pankreas und anderer peripherer Organe dienen. ...
... Auf den Anstieg der A-SMase-Aktivität folgt ein signifikanter Abfall innerhalb der ersten 24Stunden nach Beginn des Entzugs. Zum Ende des Entzugs kommt es allerdings zu einem erneuten sekundären Anstieg der A-SMase Aktivität(Reichel et al., 2010). Auch Analysen der sekretorischen A-SMase zeigten einen signifikanten Anstieg im Blutplasma bei Patienten mit Alkoholabhängigkeit. Bei diesen Patienten reduzierte sich die Aktivität der A-SMase während des Entzugs und sie erreichten schließlich eine Normalisierung der Enzymaktivität(Reichel et al., 2011). ...
... Zudem könnte der daraus resultierende erhöhte Ceramidspiegel eine alkoholinduzierte Major-Depression fördern. Daraus ergibt sich die Vermutung, dass die Aktivierung der A-SMase eine Verbindung zwischen Alkoholismus und anderen psychiatrischen Krankheiten sein könnte(Reichel et al., 2010).Ethanol führt zur Stressantwort in Gehirnzellen(Unno et al., 2002) und aktiviert sowohl die A-SMase als auch die N-SMase(Pascual et al., 2003). Durch bestimmte pathologische Prozesse kommt es zu Veränderungen des Ceramidmusters im Gehirn. ...
Die Alkoholabhängigkeit ist eine chronisch wiederkehrende Erkrankung mit hoher Prävalenz und hohem medizinischen, sozialen und wirtschaftlichen Stellenwert. Alkoholiker weisen unter anderem Defizite beim Lernen und bei der Gedächtnisbildung auf, was auf eine Dysfunktion des Hippocampus hindeutet. Man geht inzwischen davon aus, dass es im Hippocampus auch postnatal zur Neuroneogenese kommt. Kürzlich rückt zudem der Sphingolipid-Stoffwechsel bei der Erforschung der Alkoholabhängigkeit zunehmend in den Fokus. Durch die neutrale Sphingomyelinase (N-SMase) erfolgt die hydrolytische Spaltung von Sphingomyelin zu Ceramid und Phosphorylcholin. Der Konsum von Alkohol (EtOH) führt zur Stressantwort in Gehirnzellen, aktiviert die N-SMase und fördert durch die daraus resultierende Ceramidbildung den programmierten Zelltod von Nervenzellen. In dieser Arbeit untersuchen wir, welche Rolle die neutrale Sphingomyelinase bei der hippocampalen Neuroneogenese in der Maus spielt. Beim Trinkversuch wurden vier Monate alte weibliche N-SMase 2 heterozygote Knockout-Mäuse (fro/wt) (n = 8) sowie weibliche Wildtyp-Mäuse (wt/wt) (n = 8) getestet. Beide Gruppen wurden nochmal in zwei gleichgroße Gruppen, eine Expositions- und eine Kontrollgruppe (jeweils n = 4), unterteilt. Die Kontrollgruppe trank Wasser, während für die Expositionsgruppe das zwei-Flaschen Wahl Paradigma (engl.: „two-bottle free choice drinking paradigm“) herangezogen wurde. Nach einer sechswöchigen Trinkstudie, bei der die Tiere der Expositionsgruppe eine Alkoholkonzentration von 16 Vol % zu sich nahmen, wurden die Versuchstiere getötet, die Gehirne entnommen und anschließend koronare Gehirnschnitte der medialen und kaudalen Bereiche des dorsalen Hippocampus angefertigt. Die Schnitte wurden auf Nestin, Minichromosome maintenance protein 2 (MCM2) und Doublecortin (DCX) immungefärbt. Die Detektion der angefärbten Zellen in der subgranulären Zone des Gyrus dentatus erfolgte mittels Fluoreszenzmikroskopie. Insgesamt weisen die Ergebnisse darauf hin, dass der Genotyp keinen Effekt auf die adulte Neuroneogenese hat. Im Hinblick auf den Behandlungseffekt ließ sich eruieren, dass moderater Alkoholkonsum in Abhängigkeit vom Entwicklungsstadium der Neuroneogenese, moderate Effekte auf die adulte Neubildung von Nervenzellen hat. Bei den radialen Nestin-positiven/MCM2-positiven (Nestin+/MCM2+-) Stammzellen und bei den DCX-positiven (DCX+-) Neuronen zeigt sich bei den Wt-EtOH-Tieren eine signifikant geringere Dichte an markierten Zellen im Vergleich zu den Wt-Wasser-Tieren, während sowohl der Stammzellpool als auch die Gesamtzahl der proliferierenden Zellen keine Unterschiede zwischen den Gruppen vorweisen. Zudem zeigt sich ein Interaktionseffekt, den man ebenfalls bei den radialen Nestin+/MCM2+-Zellen und den DCX+-Zellen am deutlichsten sieht, während die Nestin- und MCM2-Einzelfärbungen ohne Effekt bleiben. Während im Vergleich zur Wasserbehandlung die Ethanolbehandlung bei den Wt-Tieren zur Reduktion der Neuroneogenese führt, ist die Neuroneogenese bei den Fro-Tieren in der EtOH-Gruppe im Vergleich zur Wasser-Gruppe gesteigert. Die weiblichen hetKO-Mäuse, die freiwillig moderate Mengen an Alkohol zu sich nahmen, scheinen vor der alkoholinduzierten Hemmung der adulten Neuroneogenese im Gyrus dentatus des Hippocampus geschützt zu sein. Die verminderte neuronale Apoptose ist möglicherweise auf die N-SMase 2-Hypoaktivität und der daraus resultierenden Reduktion der Bildung von Ceramid zurückzuführen. Zudem ist es möglich, dass das Ceramid, das durch die N-SMase 2 an der zytosolischen Seite der Plasmamembran gebildet wird, zum anti-apoptotischen Sphingosin-1-Phosphat umgewandelt wird. Durch welchen Mechanismus die reduzierte Aktivität der N-SMase 2 die Effekte von Alkohol moduliert, ist allerdings bis zum jetzigen Zeitpunkt unklar. Bisher gibt es keine kausale Therapiemöglichkeit für die Alkoholabhängigkeit. Die Ergebnisse dieser Arbeit weisen darauf hin, dass die N-SMase 2 eine mögliche Zielstruktur für die medikamentöse Therapie der Alkoholsucht darstellen könnte. Es bedarf weiterer Untersuchungen, um eine abschließende Aussage darüber zu treffen.
... Soy protein consumption can reduce the ceramide content of the heart, probably by reducing the expression of serine palmitoyl transferase1 (SPT-1), a key enzyme in the formation of ceramide (Torre-Villalvazo et al., 2009). Alcohol enhances the activity of ASM and results in increased ceramide levels in cell culture (Pascual et al., 2003; Saito et al., 2005), rodent models (Saito et al., 2010; Liangpunsakul et al., 2012), and humans (Reichel et al., 2010; Reichel et al., 2011). There have been mixed results with the effects of physical activity on tissue ceramide abundance in rodents (Dobrzyn et al., 2004; Helge et al., 2004; Baranowski et al., 2008; Blachnio-Zabielska et al., 2008; Tsalouhidou et al., 2009; Jung and Kang, 2010; Borg et al., 2012). ...
... Muscle ceramide has also been found to be reduced after physical exercise (Dube et al., 2011). Conversely, alcohol dependence is associated with increased peripheral lysosomal and secreted ASM activity (Reichel et al., 2010Reichel et al., , 2011) and higher concentrations of several ceramide species (Reichel et al., 2015). Oxidative stress plays an important role in the enzymatic and non-enzymatic metabolism of brain lipids (Assies et al., 2014 ). ...
... In human studies, ASM activity was increased in blood cells of intoxicated alcohol-dependent patients (Reichel et al., 2010 ). Further evidence for alcohol-induced activation of the ASM/ceramide system came from the analysis of secretory ASM in blood plasma (Kornhuber et al., 2015 ), where alcohol-dependent patients exhibited increased ASM activity in comparison to healthy controls (Reichel et al., 2011; Mühle et al., 2014 ). ...
Psychiatric disorders like mood disorders, schizophrenia, or drug addiction affect a sizeable proportion of the human population and severely compromise quality of life. Therefore, measures to prevent the manifestation, and treatments to ameliorate the symptoms, of these disorders are in high demand. Brain lipids determine the localization and function of proteins in the cell membrane of neurons. Lipids may also act as neurotransmitters or other signalling molecules. The lipid composition of the brain can be influenced by nutrition, environmental factors, and by behavioural activity. Thus, lipids represent a target for preventive medicine of psychiatric disorders. Here we review how brain lipids contribute to normal behaviour and to major psychiatric disorders with the focus on phospholipids/fatty acids, sphingolipids, and endocannabinoids. Accumulating evidence suggests a crucial role for membrane forming and signalling lipids in the brain in the etiopathologies of depression, bipolar disorders, schizophrenia, and drug addiction. Lipids also represent potential preventive interventions for these psychiatric disorders by either targeted dietary supplementation or pharmacological manipulation of lipid regulating enzymes.
... Work performed during the past decade established that ethanol induces the activation of acid and neutral sphingomyelinases (SMases) in vitro and in vivo [7,11,25,26,32,37,40,43,44,46]. SMases hydrolyze the abundant plasma membrane lipid sphingomyelin (SM) into ceramide (Cer) and phosphorylcholine. ...
... In our previous work, we showed that acute and chronic alcohol consumption leads to the activation of acid SMase (ASM) in alcoholdependent patients [43,44]. The activity of the secreted form of ASM (S-ASM) correlated positively with HDL cholesterol and other clinical markers of alcohol abuse [44]. ...
... For some analyses, patients were subgrouped according to the presence of self-reported hepatic disease (eight patients with and 11 patients without hepatic disease; four patients did not answer the question). For some analyses, patients were grouped according to their blood alcohol concentration (BAC) upon admission to the detoxification unit [43]. Eleven patients who arrived with detectable BACs were classified as 'intoxicated' and compared to already sober patients (n = 12). ...
... Serum or plasma ceramides constitute evident disease biomarkers in neurodegenerative disorders [23], obesity and diabetes mellitus [24] as well as in acute phase reactions [25], whereas their functional antagonist sphingosine1-phosphate (S1P), which promotes proliferation and cell growth, is implicated as a novel biomarker in various cancer diseases [26,27]. Similar biomarker properties such as prediction of the progression and severity of diseases like major depression [28], acute and chronic alcohol abuse [29], chronic heart failure [30], severe sepsis [31] and type 2 diabetes [32] have been attributed to ASM as well. However, despite the fact that experimental evidence has already highlighted the ASM-mediated generation of ceramide as an important mechanism mediating liver injury [19,[33][34][35][36][37], the role of SL metabolites, especially ceramide and ASM, as potential biomarkers in chronic liver disease has not been examined so far. ...
... In the present study we observed a significant upregulation of ASM activity in patients chronically infected with HCV as compared both to healthy individuals as well as to NAFLD patients (Fig. 1). The activity of ASM correlated with biochemical markers of hepatic injury, whereas demographic patient characteristics such as age, sex and BMI did not affect ASM activity levels ( Table 2), which is in line with previously published observations [28,29]. Furthermore, since serum total protein levels differed significantly between healthy individuals and patients with chronic liver disease (Table 1), we evaluated differences in ASM activity after The diagnostic performance of ASM, and sphingosine, assessed by receiver operating characteristic (ROC) curve analysis with the estimation of correspondent areas under the curve (AUC), was similar to markers of hepatic injury both in HCV and NAFLD patients compared to healthy volunteers. ...
... Secretory ASM shares the same genetic background with lysosomal ASM and gets secreted extracellularly via differential protein trafficking by endothelial cells, skin fibroblasts, monocytes and macrophages [41]. In this context, an increased activity of serum ASM has been identified both in immune-mediated inflammation [30,31,42] as well as in liver injury caused by copper [19] and alcohol [29,43], while ASM has been recently suggested as a key player in liver fibrosis by interacting with cathepsins B/D [44]. Catalytic activation of the ASM occurs either via protein kinase Cδ (PKCδ)-mediated post-translational modification of the enzyme turnover [45] or, alternatively, from increased transcriptional regulation of the ASM gene [46]. ...
Sphingolipids constitute bioactive molecules with functional implications in homeostasis and pathogenesis of various diseases. However, the role of sphingolipids as possible disease biomarkers in chronic liver disease remains largely unexplored. In the present study we used mass spectrometry and spectrofluorometry methods in order to quantify various sphingolipid metabolites and also assess the activity of an important corresponding regulating enzyme in the serum of 72 healthy volunteers as compared to 69 patients with non-alcoholic fatty liver disease and 69 patients with chronic hepatitis C virus infection. Our results reveal a significant upregulation of acid sphingomyelinase in the serum of patients with chronic liver disease as compared to healthy individuals (p < 0.001). Especially in chronic hepatitis C infection acid sphingomyelinase activity correlated significantly with markers of hepatic injury (r = 0.312, p = 0.009) and showed a high discriminative power. Accumulation of various (dihydro-) ceramide species was identified in the serum of patients with non-alcoholic fatty liver disease (p < 0.001) and correlated significantly to cholesterol (r = 0.448, p < 0.001) but showed a significant accumulation in patients with normal cholesterol values as well (p < 0.001). Sphingosine, a further bioactive metabolite, was also upregulated in chronic liver disease (p < 0.001). However, no significant correlation to markers of hepatic injury was identified.
Conclusion
Chronic hepatitis C virus infection and non-alcoholic fatty liver disease induce a significant upregulation of serum acid sphingomyelinase which appears as a novel biomarker in chronic hepatopathies. Further studies are required to elucidate the potential of the sphingolipid signaling pathway as putative therapeutic target in chronic liver disease.
... Serum or plasma ceramides constitute evident disease biomarkers in neurodegenerative disorders [23], obesity and diabetes mellitus [24] as well as in acute phase reactions [25], whereas their functional antagonist sphingosine1-phosphate (S1P), which promotes proliferation and cell growth, is implicated as a novel biomarker in various cancer diseases [26,27]. Similar biomarker properties such as prediction of the progression and severity of diseases like major depression [28], acute and chronic alcohol abuse [29], chronic heart failure [30], severe sepsis [31] and type 2 diabetes [32] have been attributed to ASM as well. However, despite the fact that experimental evidence has already highlighted the ASM-mediated generation of ceramide as an important mechanism mediating liver injury [19,[33][34][35][36][37], the role of SL metabolites, especially ceramide and ASM, as potential biomarkers in chronic liver disease has not been examined so far. ...
... In the present study we observed a significant upregulation of ASM activity in patients chronically infected with HCV as compared both to healthy individuals as well as to NAFLD patients (Fig. 1). The activity of ASM correlated with biochemical markers of hepatic injury, whereas demographic patient characteristics such as age, sex and BMI did not affect ASM activity levels ( Table 2), which is in line with previously published observations [28,29]. Furthermore, since serum total protein levels differed significantly between healthy individuals and patients with chronic liver disease (Table 1), we evaluated differences in ASM activity after The diagnostic performance of ASM, and sphingosine, assessed by receiver operating characteristic (ROC) curve analysis with the estimation of correspondent areas under the curve (AUC), was similar to markers of hepatic injury both in HCV and NAFLD patients compared to healthy volunteers. ...
... Secretory ASM shares the same genetic background with lysosomal ASM and gets secreted extracellularly via differential protein trafficking by endothelial cells, skin fibroblasts, monocytes and macrophages [41]. In this context, an increased activity of serum ASM has been identified both in immune-mediated inflammation [30,31,42] as well as in liver injury caused by copper [19] and alcohol [29,43], while ASM has been recently suggested as a key player in liver fibrosis by interacting with cathepsins B/D [44]. Catalytic activation of the ASM occurs either via protein kinase Cδ (PKCδ)-mediated post-translational modification of the enzyme turnover [45] or, alternatively, from increased transcriptional regulation of the ASM gene [46]. ...
... In alcohol-dependent patients, ASM activity is increased compared to healthy controls. High levels of cellular ASM activity in peripheral blood cells was associated with acute intoxication, whereas the activity of secreted ASM (S-ASM) in blood plasma was more related to the chronic consumption of alcohol (Reichel et al. 2010. S-ASM activity declined gradually in patients that withdrew from alcohol, and it correlated with the levels of carbohydrate-deficient transferrin (CDT). ...
... Sphingomyelinase-induced ceramide generation may contribute to the emergence of complex and multifactorial psychiatric disorders (Kornhuber et al. 2009;Gulbins et al. unpublished). In this model, several stress-related factors including psychoactive substances, inflammatory cytokines, reactive oxygen species, and infections may induce activation of sphingomyelinases (Grassmé et al. 2003(Grassmé et al. , 2005Haimovitz-Friedman et al. 1997;Hofmeister et al. 1997;Nalivaeva et al. 2000;Reichel et al. 2010Reichel et al. , 2011Scheel-Toellner et al. 2004;Wong et al. 2000;Zhang et al. 2008) and result in increased levels of ceramide in various brain areas. This model may account (to varying degrees) for several psychiatric disorders besides depression (Table 1). ...
... Depression ASM activity " in peripheral blood cells of patients Kornhuber et al. (2005) Cer " in plasma levels of patients altered plasma levels of SM and PC species in patients Cer " in mouse model Deaciuc et al. (2000), Liangpunsakul et al. (2010Liangpunsakul et al. ( , 2012 ASM activity " in cell culture experiment Pascual et al. (2003), Liu et al. (2000) ASM activity " in mouse model Deaciuc et al. (2000) ASM activity " in patients Reichel et al. (2010Reichel et al. ( , 2011 NSM activity " in cell culture experiment Pascual et al. (2003), Liu et al. (2000) Anxiety S1P ...
Despite the high prevalence and devastating impact of psychiatric disorders, little is known about their etiopathology. In this review, we provide an overview on the participation of sphingolipids and enzymes responsible for their metabolism in mechanisms underlying psychiatric disorders. We focus on the pathway from sphingomyelin to proapoptotic ceramide and the subsequent metabolism of ceramide to sphingosine, which is in turn phosphorylated to yield anti-apoptotic sphingosine-1-phosphate (S1P).
... 17 Enzyme activity is dynamically regulated also in peripheral tissue. In the blood, it may serve as a biomarker for alcohol consumption 18,19 and a depressive state. 20,21 A particular role of sphingolipid changes in the brain was reported for extinction learning, when a naturally reinforced operant behavior was no longer rewarded and behavioral responses had to be adapted. ...
... Chronic alcohol exposure was associated with increased peripheral ASM activity 18 and higher concentrations of several ceramide species in humans. 52 The observed opposite direction of effects for cocaine may reflect the distinct ways of action of the different classes of drugs. ...
Cocaine addiction is a severe psychiatric condition for which currently no effective pharmacotherapy is available. Brain mechanisms for the establishment of addiction‐related behaviors are still not fully understood, and specific biomarkers for cocaine use are not available. Sphingolipids are major membrane lipids, which shape neuronal membrane composition and dynamics in the brain. Here, we investigated how chronic cocaine exposure during establishment of addiction‐related behaviors affects the activity of the sphingolipid rheostat controlling enzymes in the brain of rats. As we detected specific effects on several enzymes in the brain, we tested whether the activity of selected enzymes in the blood may serve as potential biomarker for cocaine exposure in non‐human primates (Callithrix penicillata ). We found that intravenous cocaine self‐administration led to a reduced mRNA expression of Cers1 , Degs1 and Degs2 , and Smpd1 in the prefrontal cortex of rats, as well as a reduction of Cers4 expression in the striatum. These effects reversed after 10 days of abstinence. Monkeys showed a robust cocaine‐induced place preference (CPP). This coincided with a reduction in blood acid sphingomyelinase (ASM) activity after CPP establishment. This effect normalized after 15 days of abstinence. Altogether, these findings suggest that the establishment of cocaine addiction‐related behaviors coincides with changes in the activity of sphingolipid controlling enzymes. In particular, blood ASM levels may serve as a translational biomarker for recent cocaine exposure.
... Recent studies have observed that ceramide, one of the crucial sphingolipids in biological membranes, might be link between depression and alcohol use disorder. Clinical studies showed a significant increase in the level of lysosomal and secretory acid sphingomyelinase (ASM) in peripheral blood cells of patients with acute alcohol intoxication (10)(11)(12)(13). Similarly, a rise in the concentrations of total ceramide as well as the products of ceramide metabolism, sphingosine and sphinganine, was observed in the liver of alcohol-fed mice (14).The levels of ceramide precursor sphingomyelin is found to be reduced in the blood serum of rats exposed to liquid alcohol-containing diet as well as in alcohol dependent patients (15). ...
... Previous data indicate the contribution of Asm in alcohol sensitivity and pathogenesis of alcohol use disorder (10,11,14,15,27). Here, we report the higher preference of alcohol on the model of free-choice alcohol drinking in Asm−/− mice compared to wt littermates. ...
Major depression and alcohol use disorder are severe psychiatric diseases affecting the world's population with high comorbidity level. However, the pathogenesis of this comorbidity remains unclear, and no selective treatment for this condition is available. A pathogenic pathway and a possible therapeutic target for the treatment of depression-alcoholism comorbidity based on the hyperfunction of acid sphingomyelinase (Asm) were recently suggested. Here we analyzed the effects of alcohol on the depression/anxiety state of homozygous Asm-knockout mice (Asm − /−), which can be considered as a model of an early stage of Niemann-Pick disease, as well as their drinking pattern under normal and stress conditions. It was observed that forced treatment with alcohol (2 g/kg, i.p.) reduces the anxiety level of Asm−/− mice as measured in the elevated plus maze (EPM) test, but enhances the depression level in the forced swim test (FST). The analysis of drinking pattern of these animals in a free-choice alcohol drinking paradigm revealed higher alcohol intake and preference in Asm−/− mice compared to wild type (wt) littermates. However, this difference was overwritten by the stress exposure. Stronger sedating effects of alcohol were observed in Asm−/− mice compared to wt animals in the loss of righting reflex test after single and repeated alcohol injections (3 g/kg, i.p.). Altogether, the present findings might indicate an Asm involvement in the mechanisms of comorbidity between alcoholism and anxiety/depression.
... MDD and alcohol dependence share a high comorbidity, which can partially be explained by the use of alcohol as a way of self-medication for psychiatric problems, including depression. In alcohol dependence, the activity of L-ASM in peripheral mononuclear blood cells is significantly increased and decreases during withdrawal treatment [27]. The same effect is considerably larger for the secretory form of the enzyme [28][29][30]. ...
... The absence of an increase is in contrast to the stronger response of S-ASM (3-fold higher levels than controls [28]) compared to L-ASM [27] to chronic alcohol consumption, suggesting that S-ASM could respond more sensitively to physiological alterations or at least to alcohol. The increased levels of plasma S-ASM (by 60%) in 11 combat veterans with post-traumatic stress disorder compared to five male controls [23] might be specific to this disease entity. ...
Major depressive disorder (MDD) is a highly prevalent and devastating psychiatric illness with strong individual and societal burdens. However, biomarkers to improve the limited preventive and therapeutic approaches are scarce. Multilevel evidence suggests that the pathophysiological involvement of sphingolipids particularly increases the levels of ceramides and the ceramide hydrolyzing enzyme, acid sphingomyelinase. The activity of secretory acid sphingomyelinase (S-ASM) and routine blood parameters were determined in the serum of patients with current (unmedicated n = 63, medicated n = 66) and remitted (n = 39) MDD and healthy subjects (n = 61). Depression severity and anxiety and their 3-weeks prospective course of treatment were assessed by psychometric inventories. S-ASM activity was not different between the four groups, did not decrease during treatment, and was not lower in individuals taking medication that functionally inhibited ASM. However, S-ASM correlated positively with depression severity only in remitted patients. High enzyme activity at inclusion predicted milder clinician-evaluated and self-rated depression severity (HAM-D, MADRS, BDI-II) and state anxiety at follow-up, and was related to stronger improvement in these scores in medicated patients. S-ASM was strongly and contrariwise associated with serum lipids in unmedicated and medicated females. These findings contribute to a better understanding of the pathomechanisms underlying depression and the development of clinical strategies and biomarkers.
... Activation of ASM and a concomitant increase in ceramide concentrations can be induced by the stimulation of various receptors, including CD95 or those for tumor necrosis factor (TNF)-α, interleukin (IL)-1β or platelet-activating factor, or by cellular stress such as inflammation, infection, ischemia, radiation, oxidative stress, chemotherapeutic agents or cell wounding [3][4][5][6][7][8][9][10][11][12]. Increased ASM activity was observed in several human diseases such as chronic heart failure, type 2 diabetes, sepsis, peritonitis, hepatitis, hemophagocytic lymphohistiocytosis, antineutrophil cytoplasmic antibody-associated primary systemic vasculitis, neurodegeneration, depression and alcohol abuse [13][14][15][16][17][18][19][20][21][22][23]. A variety of small organic, drug-like compounds that functionally inhibit ASM are used in the therapy of human diseases [24][25][26]. ...
... Determination of ASM activity S-ASM and L-ASM activities were assessed from blood plasma and peripheral blood mononuclear cells respectively, as previously described [21,22]. ...
Background: Acid sphingomyelinase (ASM) is a key regulator of ceramide-dependent signalling pathways. Among others, activation of ASM can be induced by CD95 or cytokine signalling and by cellular stress resulting from inflammation or infection. Increased ASM activity was observed in a variety of human diseases including inflammatory and neuropsychiatric disorders. We hypothesized that basal ASM activity might influence the susceptibility for common human diseases. Methods: The general health condition of 100 young people was assessed using a questionnaire. The ASM polymorphism rs1050239 (c.1522G>A; encoding p.G508R) was determined from genomic DNA. Activities of secretory (S-) and lysosomal (L-) ASM were measured in blood plasma and peripheral blood cells respectively. Results: The polymorphism rs1050239 was significantly associated with self-reported allergy (p=4.68×10(-4); adjusted p-value for multiple testing 0.007). Allergy was more prevalent in carriers of the minor A allele compared to non-carriers (p=0.00015; odds ratio=6.5, 95% CI 2.15-21.7). S-ASM activity was significantly associated with rs1050239 (p=5.3×10(-7)) and decreased with the number of A alleles in a gene-dosage dependent manner. In allergic patients, S-ASM activity was moderately decreased (p=0.034). L-ASM activity was significantly lower in subjects homozygous for the minor A allele (p=0.025) but not different between allergic and non-allergic subjects (p=0.318). Conclusion: Our analysis provides evidence for an involvement of ASM in the pathophysiology of allergy, which is in line with previous reports addressing the role of sphingolipids in this disorder. Further studies should clarify the mechanism linking rs1050239 to allergy. The ASM pathway might be useful for predicting allergic disposition and disease course and as a therapeutic target. © 2014 S. Karger AG, Basel.
... Accumulation of SM predominates in the viscera (Niemann-Pick Disease type B) or can extend to neuronal and glial cells resulting in cognitive deficits, motor dysfunction and blindness (type A). Increased ASM levels have been implicated in various pathological conditions including atheroscle-rosis [9], major depression [10], Alzheimer's disease [11], status epilepticus [12] and alcoholism [13,14]. ...
... Assays for lysosomal ASM activity in cell or tissue lysates are well-established using radioactive 14 C or fluorescent substrates with reaction times of minutes to hours. In a recent report on SASM in plasma [13], incubation with BODIPY-C12-SM for 24 h resulted in strong signals. ...
As a key enzyme in sphingolipid metabolism, acid sphingomyelinase (ASM) is involved in the regulation of cell fate and signaling via hydrolysis of sphingomyelin to form ceramide. While increased activity of the lysosomal form has been associated with various pathological conditions, there are few studies on secretory ASM limited only to cell models, plasma or serum.
An optimized assay based on a fluorescent substrate was applied to measure the ASM activity in cerebrospinal fluid (CSF) collected from mice and from 42 patients who were classified as controls based on normal routine CSF values.
We have detected ASM activity in human CSF, established a sensitive quantitative assay and characterized the enzyme's properties. The enzyme resembles plasmatic ASM including protein stability and Zn(2+)-dependence but the assays differ considerably in the optimal detergent concentration. Significantly increased activities in the CSF of ASM transgenic mice and undetectable levels in ASM knock-out mice prove that the measured ASM activity originates from the ASM-encoding gene SMPD1. CSF localized ASM activities were comparable to corresponding serum ASM levels at their respective optimal reaction conditions, but no correlation was observed. The large variance in ASM activity was independent of sex, age or analyzed routine CSF parameters.
Human and mouse CSF contain detectable levels of secretory ASM, which are unrelated to serum ASM activities. Further investigations in humans and in animal models will help to elucidate the role of this enzyme in human disease and to assess its value as a potential biomarker for disease type, severity, progress or therapeutic success.
... in disorders such as Alzheimer's dementia,47,48 ethanol-induced cell death in humans,45,49 stroke, and ischemia.40,50 4 | FIASMAs CAN INHIBIT SARS-CoV-2 REPLICATIONRecently, some FIASMAs (chiefly fluoxetine, a selective SSRI) have been shown to inhibit SARS-CoV-2 replication in different in vitro models.[7][8][9][10][11] ...
In the last 2 years, different pharmacological agents have been indicated as potential inhibitors of SARS-CoV-2 in vitro. Specifically, drugs termed as functional inhibitors of acid sphingomyelinase (FIASMAs) have proved to inhibit the SARS-CoV-2 replication using different types of cells. Those therapeutic agents share several chemical structure characteristics and some well-known representatives are fluoxetine, escitalopram, fluvoxamine, and others. Most of the FIASMAs are primarily used as effective therapeutic agents to treat different pathologies, therefore, they are natural drug candidates for repositioning strategy. In this review, we summarize the two main proposed mechanisms mediating acid sphingomyelinase (ASM) inhibition and how they can explain the inhibition of SARS-CoV-2 replication by FIASMAs. The first mechanism implies a disruption in the lysosomal pH fall as the endosome-lysosome moves toward the interior of the cell. In fact, changes in cholesterol levels in endosome-lysosome membranes, which are associated with ASM inhibition is thought to be mediated by lysosomal proton pump (ATP-ase) inactivation. The second mechanism involves the formation of an extracellular ceramide-rich domain, which is blocked by FIASMAs. The ceramide-rich domains are believed to facilitate the SARS-CoV-2 entrance into the host cells.
... In addition to this evidence of NSM involvement in alcohol addiction, similar proof was found for ASM: In acutely alcohol-intoxicated patients, increased lysosomal ASM was observed in peripheral blood mononuclear cells as well as in plasma, which returned to normal levels during withdrawal [328,329]. Remarkably, a correlation between secretory ASM activity and carbohydrate deficit transferrin (CDT) -a clinical biomarker for alcohol abuse -was demonstrated [329]. In summary, it can be concluded that acute and chronic alcohol consumption leads to activation of ASM, which returns to a normal level with abstinence. ...
Introduction Alcohol use disorder is a serious psychiatric disorder that poses major challenges to the health care system. The development of the disease is often multifactorial, and a minority of patients can be successfully treated with medication. Several neurotransmitter systems, particularly the serotonergic and dopaminergic reward pathways, are affected by alcohol exposure and contribute to the development of alcohol dependence. Here, the sphingolipids and their metabolism are considered mediators in the development of addictive disorders including alcohol use disorder. Indeed, increased activity of the acid sphingomyelinase (ASM), which has been identified as a key enzyme of sphingolipid metabolism, not only affects monoamine homeostasis in the brain by reducing tissue levels of serotonin (5-HT) and dopamine (DA) in the ventral striatum, but also increases alcohol intake. However, remarkably high levels of another sphingomyelinase are found in the brain: neutral sphingomyelinase 2 (NSM). Interestingly in early abstinent patients with alcohol dependence, not only an increase in NSM activity was measured. In a cell model, decreased activity of NSM causes a decrease in 5-HT uptake, DA uptake, and DA release. However, besides the reported impairment of brain monoamines, further results indicate an association between single nucleotide polymorphisms in the NSM-encoding gene Sphingomyelin phosphodiesterase (SMPD) 3 and alcohol intake. This work aimed to investigate the influence of NSM hypoexpression in mice on central monoaminergic signaling in a model of voluntary alcohol consumption. Methods 24 male and 24 female C57 black6/J mice, half of which carried a heterozygous fragilitas ossium (fro) mutation, were used for the study. In the fro mice, one allele of the gene encoding NSM was knocked out. In a two-bottle paradigm, all animals received ethanol at increasing concentrations (2-16% by volume). The control group was exposed to tap water. Alcohol consumption, alcohol preference, water intake, and total intake of all groups were determined. After 36 days of the experiment and sufficient establishment of alcohol drinking behaviour, the animals were killed, and the removed brains were examined for mRNA expression of dopamine and serotonin receptors/transporters in the ventral striatum. Results NSM hypoactivity partially increases voluntary alcohol consumption in male mice at 2% alcohol solution with no effect on the total intake. Moreover, NSM regulates monoaminergic signalling in male mice: Alcohol-induced alteration of serotonin-1a-receptor, serotonin-2c-receptor, serotonin- transporter and dopamine-transporter was prevented. This effect appears to be strongly sex dependent. With respect to female littermates, our results do not indicate a significant change in voluntary alcohol consumption or total intake. However, alcohol naive female fro mice show significantly reduced D2 expression. Alcohol consumption further reveals an effect of NSM hypoexpression on monoaminergic signalling in females: On the one hand, alcohol-induced neuroplasticity was prevented as in serotonin-3a-receptor, and on the other hand, new adaptations of other receptors such as serotonin-1a-receptor occurred. Conclusion Our results reveal the crucial influence of the fro mutation on the adaptation of monoaminergic signalling to alcohol exposure. Interestingly, the effect of NSM on the monoaminergic system turns out to be strongly sex dependent. This results in lower susceptibility to alcohol-induced neuroplasticity due to NSM hypoexpression in males. Furthermore, we report an influence of NSM hypoexpression on alcohol consumption in men, namely partially reduced alcohol intake, but not in women. These results indicate the involvement of the NSM and possible downstream mechanisms in the development of alcohol dependence and may provide a possible basis for explaining the sex difference in alcohol use disorders. Furthermore, the dopaminergic and serotoninergic reward systems are essential for the reinforcing effects of alcohol and the transition to addiction. The fact that NSM exerts an influence on the monoaminergic response to alcohol exposure supports the idea that NSM may be a promising pharmacological target for the treatment of alcohol dependence.
... But there is also evidence that alcohol exposure and withdrawal have potent effects on the activity of acid sphingomyelinase itself. Acid sphingomyelinase is upregulated in response to acute alcohol exposure and in alcohol-dependent patients, although this upregulation gradually returns to normal levels during alcohol withdrawal (Reichel et al., 2010;Reichel et al., 2011). However, both the acute alcohol-induced increase in acid sphingomyelinase, and the withdrawal-induced decrease in the same, are greater in males (Mühle et al., 2014), suggesting possible sex-dependent effects in related diseases. ...
Despite the prevalence and well-recognized adverse effects of prenatal alcohol exposure and alcohol use disorder in the causation of numerous diseases, their potential roles in the etiology of neurodegenerative diseases remain poorly characterized. This is especially true of the rare neurodegenerative diseases, for which small population sizes make it difficult to conduct broad studies of specific etiological factors. Nonetheless, alcohol has potent and long-lasting effects on neurodegenerative substrates, at both the cellular and systems levels. This review highlights the general effects of alcohol in the brain that contribute to neurodegeneration across diseases, and then focuses on specific diseases in which alcohol exposure is likely to play a major role. These specific diseases include dementias (alcohol-induced, frontotemporal, and Korsakoff syndrome), ataxias (cerebellar and frontal), and Niemann-Pick disease (primarily a Type B variant and Type C). We conclude that there is ample evidence to support a role of alcohol abuse in the etiology of these diseases, but more work is needed to identify the primary mechanisms of alcohol’s effects.
... The post hoc analysis revealed a higher percentage of lymphocytes in severely depressed patients (36.6% ± 10.1) compared with moderately depressed patients (31.2% ± 6.5, Tukey, p < 0.05) and healthy participants (31.6% ± 7.8, Tukey, p = 0.06). To rule out a confounding effect of heterogeneity of the sample composition [40], we calculated the SMPD1 mRNA expression per percentage of lymphocytes. ANOVA revealed a trend towards statistical significance regarding an increased level of SMPD1 mRNA expression in our sample of depressed patients (F(2, 118) = 2.39, p = 0.10). ...
Major depressive disorder (MDD) is a severe psychiatric condition with key symptoms of low mood and lack of motivation, joy, and pleasure. Recently, the acid sphingomyelinase (ASM)/ceramide system has been implicated in the pathogenesis of MDD. ASM is a lysosomal glycoprotein that catalyzes the hydrolysis of sphingomyelin, an abundant component of membranes, into the bioactive sphingolipid ceramide, which impacts signaling pathways. ASM activity is inhibited by several common antidepressant drugs. Human and murine studies have confirmed that increased ASM activity and ceramide levels are correlated with MDD. To define a molecular marker for treatment monitoring, we investigated the mRNA expression of SMPD1, which encodes ASM, in primary cell culture models, a mouse study, and a human study with untreated MDD patients before and after antidepressive treatment. Our cell culture study showed that a common antidepressant inhibited ASM activity at the enzymatic level and also at the transcriptional level. In a genetically modified mouse line with depressive-like behavior, Smpd1 mRNA expression in dorsal hippocampal tissue was significantly decreased after treatment with a common antidepressant. The large human study showed that SMPD1 mRNA expression in untreated MDD patients decreased significantly after antidepressive treatment. This translational study shows that SMPD1 mRNA expression could serve as a molecular marker for treatment and adherence monitoring of MDD.
... Activity of ASM is sensitive to cellular stress and is activated, among others, by TNF-α (22), oxidative stress (23), and ionizing radiation (24). In line with these findings, recent studies provided evidence that both stress-associated disorders, as for instance major depression (25), chronic heart failure (26), acute and chronic alcohol consumption (27,28), and chronic hepatitis C infection (29), as well as various chronic unpredictable stressors (25,30), have been associated with increased ASM activity and Cer levels, respectively. ...
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.
... The other reason for identifying causes of high haemolysis is, of course, to use the information to make general improvements to the RBC storage quality; it is possible that information about sphingomyelins and sphingomyelin metabolism could be employed in this regard. The amounts of some sphingolipids present can be increased if they are collected from fasting subjects [12]; sphingomyelinase activity is higher in the RBCs of people who drink more alcohol [19,20]; the activity of sphingomyelinase has been characterised [21] and could possibly be inhibited; the formation of ceramides and ceramide-related RBC death has also be characterised, along with possible inhibitors [17]; ceramide domains have been associated with haemolysis occurring after cooling [22]. A ceramide-mediated haemolysis could also account for the improved RBC storage in isotonic PAGGSM relative to the storage in the hypertonic SAGM [23]: the formation of ceramides is linked to the osmotically induced cell shrinkages [17], and so the lower osmolarity of the PAGGSM could therefore account for its improvement of the RBC storage. ...
Background and objectives
Red blood cells that are stored for transfusions as red cell concentrates (RCCs) undergo changes during the storage period, culminating in the lysis of the cells. The goal of this work is to find markers that are linked to high haemolysis, in order to explain the inter‐donor variability that is known to occur in storage quality, and also the known differences between RCCs from male and female donors.
Materials and methods
The relative amounts of lipids at the end of the storage period were compared for one group of low haemolysis samples (24 units, all ≤0·15% haemolysis), and one group of high haemolysis samples (26 units, all ≥0·5% haemolysis). Representative lipids were analysed from different lipid classes, including cholesterol, phosphatidylcholine, phosphatidylethanolamine, sphingomyelin and ceramide. Whole membrane preparations were analysed with one mass spectrometry technique, and lipid extracts were analysed with a second mass spectrometry technique.
Results
The ratio of palmitoyl‐oleoyl phosphatidylcholine (POPC) to sphingomyelin was different for the high and low haemolysis groups (P = 0·0001) and for the RCCs from male and female donors (P = 0·0009). The ratio of cholesterol to phospholipids showed only minimal links to haemolysis. Higher relative amounts of sphingomyelin were associated with lower haemolysis, and higher relative amounts of ceramides were associated with increased haemolysis.
Conclusion
The level of sphingomyelinase activity and the resulting ratio of sphingomyelin to POPC is proposed as a possible marker for RCC storage quality.
... Rodent models of alcohol consumption have revealed similar findings to human studies with increased ASMase activity in mouse and rat tissues after exposure to ethanol [50,51]. In rats, addition of N-acetylcysteine to the ethanol treatment reduced ASMase activity, steatosis and inflammation, implicating oxidative stress as one of the drivers of the enzyme activation. ...
Epidemiological evidence underscores alcohol consumption as a strong risk factor for multiple cancer types, with liver cancer being most commonly associated with alcohol intake. While mechanisms linking alcohol consumption to malignant tumors development are not fully understood, the likely players in ethanol-induced carcinogenesis are genotoxic stress caused by formation of acetaldehyde, increased oxidative stress and altered nutrient metabolism, including the impairment of methyl transfer reactions. Alterations of sphingolipid metabolism and associated signaling pathways are another potential link between ethanol and cancer development. In particular, ceramides are involved in the regulation of cellular proliferation, differentiation, senescence and apoptosis and are known to function as important regulators of malignant transformation as well as tumor progression. However, to date, the cross-talk between ceramides and alcohol in cancer disease is largely an open question and only limited data are available on this subject. Most studies linking ceramide to cancer considered liver steatosis as the underlying mechanism, which is not surprising taking into consideration that ceramide pathways are an integral part of the overall lipid metabolism. This review summarizes the latest studies pointing to ceramide as important mediator of cancer-promoting effects of chronic alcohol consumption and underscores the necessity of understanding the role of sphingolipids and lipid signaling in response to alcohol in order to prevent and/or successfully manage diseases caused by alcohol.
... 4,5 As such, increased ASM activity was found in peripheral blood mononuclear cells of patients experiencing a major depressive episode 6 and in acutely intoxicated alcoholdependent patients. 7 ASM activity was also increased in plasma and serum of alcohol-dependent patients, 8,9 where it correlated positively with blood alcohol concentrations. Patients experiencing a major depressive episode during the past 2 years showed increased plasma levels of several ceramide species including C16:0, C18:0, C20:0, C24:1, and C26:1, but not C22:0 or C24:0, compared with both healthy controls and patients experiencing a depressive episode more than 2 years ago. ...
Depression and alcohol dependence are associated with increased plasma ceramide concentrations in humans. Pharmacological increase in C16 ceramide concentrations in the dorsal hippocampus (DH) induced a depressive-like phenotype in naïve mice. However, the effects of C16 ceramide on alcohol consumption and anxiety-like behavior as well as the behavioral effects of other ceramide species are yet unknown. Therefore, we investigated whether repeated infusion of ceramides with different fatty acid chain lengths (C8, C16, and C20) into the DH and the basolateral amygdala (BLA) alter alcohol consumption, emotional behavior, and tissue monoamine levels. Our results revealed that C16, but not C8 and C20, ceramide altered alcohol drinking and emotional behavior in a brain region-specific way without altering tissue noradrenaline, dopamine, and serotonin levels in the prefrontal cortex, ventral striatum, and dorsal mesencephalon. In more detail, C16 ceramide increased alcohol consumption when infused into the BLA, but not when infused into the DH. Furthermore, C16 ceramide induced a depressive-like phenotype when infused into the DH, but a predominantly anxiogenic-like phenotype (in a non-social, but not a social context) when infused into the BLA. In turn, alcohol drinking normalized C16 ceramide-induced depressive-like and anxiogenic-like phenotypes. This study demonstrates a complex ceramide species-specific and brain region-specific modulation of alcohol consumption and emotional behavior in mice and provides the framework for future studies investigating the involvement of distinct ceramide species in the regulation of emotional behavior.
... Aufgrund des in Vorversuchen gezeigten Anstiegs von ASM, NSM [126][127][128] [70]. Es ist bereits bewiesen, dass die AC und NC von Endothelzellen aktiv sezerniert werden [132,133]. ...
Hintergrund und Ziele: Der Großteil aller Lipide einer Zelle sind Sphingolipide. Im Stoffwechsel dieser Mole-külgruppe nimmt die Ceramidase die Funktion eines Schlüsselenzyms ein. Sie katalysiert die Hydrolyse von Ceramid zu Sphingosin und freier Fettsäure. Eine Anhäufung von Ceramid in der Zelle wirkt zelltoxisch und führt zur Apoptose. Ein Mangel an Ceramidase kann so zu Erkrankungen, wie zum Beispiel der Farber-Krankheit, führen. Bei Alkoholabhängigkeit ist der Sphingolipidstoffwechsel verändert: In Zellen wie im Plasma alkoholabhängiger Patienten finden sich erhöhte Aktivitäten der sauren Sphin-gomyelinase, einem Enzym, das Ceramid produziert. Im Zellkulturmodell wurden eben-falls Veränderungen des Ceramidspiegels nach Behandlung mit Ethanol beschrieben. Daher sollte anhand eines Rattenmodells die Auswirkung von Ethanolkonsum auf die Ceramidaseaktivität im Gewebe und Plasma untersucht werden. Methoden Adulte Wistarratten wurden in 4 Versuchsgruppen à 6 Tiere aufgeteilt und für 4 Wo-chen behandelt. Gruppe 1 erhielt als Trinkflüssigkeit Wasser (Kontrollgruppe), Gruppe 2 erhielt ebenfalls Wasser, vor der Dekapitation wurde allerdings ein Wasserbolus über eine Schlundsonde verabreicht, um einen Stressreiz zu simulieren. Die Versuchsgruppen 3 und 4 erhielten Ethanol ad libitum (chronischer Alkoholkonsum). In Gruppe 4 wurde vor der Dekapitation ein Ethanolbolus verabreicht, um akuten Alkoholkonsum vor dem Hintergrund einer Alkoholabhängigkeit zu simulieren. Nach der Dekapitation wurden den Ratten Leber, Lunge, Milz, Striatum, Hippocampus, und Präfrontalem Cortex (PFC) sowie Blut zur Plasmagewinnung entnommen. Die Ceramidase-Aktivität im sauren (AC) und neutralen (NC) pH-Bereich wurde anhand der Hydrolyse von fluoreszenzmarkiertem NBD-C12-Ceramid und Trennung der Hydrolyseprodukte NBD-C-12-markierte Fettsäure und Sphingosin mittels Dünnschichtchromatographie quantifiziert. Neben dem Vergleich der Ceramidase-Aktivität in den Organen und im Plasma wurden Korrelationen der Enzymaktivitäten untereinander sowie mit den Haltungsdaten (Körpergewicht, Kalorienaufnahme, etc.) untersucht. Ergebnisse und Beobachtungen: Insgesamt wurden nur wenige signifikante Effekte deutlich. Die Aktivität der NC in der Leber nach akutem und chronischem Ethanolkonsum war im Vergleich zur Kontroll-gruppe signifikant erniedrigt. Eine verminderte Ceramidaseaktivität könnte eine Anhäufung des zelltoxischen Ceramids zur Folge haben und damit die zellschädigende Wirkung von Ethanol erklären. Im Plasma derselben Versuchsgruppe (Gruppe 4) zeigte sich eine erhöhte Aktivität der NC im Plasma. Dies deckt sich mit dem bekannten Anstieg der sauren Sphingomyelinase-Aktivität im Plasma bei akut alkoholintoxikierten Alkoholabhängigen. Darüber hinaus fand sich eine erniedrigte Aktivität der AC im Hippocampus durch den Stressreiz in Gruppe 2 und könnte auf eine schnelle Stressregulation der Ceramidase hindeuten. Bemerkenswert war auch der Zusammenhang zwischen der Aktivität der Ceramidasen und Gewichtsparametern, welche in allen Gruppen auftraten, allerdings nicht spezifisch für ein Gewebe waren. Besonders auffällig war die sehr hohe Varianz der Leber-Enzymaktivität mit hohen versus niedrigen Werten in je etwa der Hälfte aller Tiere, deren Ursache noch unbekannt blieb. Zusätzlich zeigte sich eine besonders im neuropsychiatrischen Kontext interessante Verteilung der AC und der NC im Gewebe der Kontrolltiere: eine hohe Aktivität beider Enzyme in verschiedenen proteinärmeren Hirnregionen gegenüber einer niedrigeren Aktivität in den proteinreicheren peripheren Organen. Schlussfolgerungen: Eine Herabregulation bzw. eine fehlende Hochregulation der Ceramidaseaktivität paral-lel zu einer erhöhten Sphingomyelinaseaktivität nach Ethanolkonsum könnte zu einer Anhäufung des zelltoxischen Ceramids und einer Verschiebung des Rheostats der Zelle in Richtung Apoptose führen. Die genauen Abläufe innerhalb der Zelle sind noch nicht geklärt und sollten weiter untersucht werden. Unter anderem wären kontinuierliche Blutuntersuchungen im Versuchsverlauf interessant. Sinnvoll wären zudem Versuche an Mäusen mit höherem erreichbaren Alkoholkonsum, mit größerer Gruppenzahl und beiden Geschlechtern um zuverlässige geschlechtsspezifische Effekte zu erkennen. Zusätz-lich wäre die Untersuchung von weiblichen Ratten aufschlussreich, um den bekannten Geschlechtseffekt der Ceramidase im Blut auch an Organen untersuchen zu können. Die Aktivierung der Ceramidase würde neue Möglichkeiten in der Behandlung von alko-holbedingten Erkrankungen und Folgen (z.B. Formen der vorgeburtlichen Schädigung durch Alkohol) eröffnen.
... Altered enzyme activities have been noted in a variety of common human diseases [12]. In alcohol dependence, levels of both the lysosomal [13] and secretory form (S-ASM) of the enzyme are increased, and decrease gradually during withdrawal treatment in male, as well as female, patients [14,15]. Consequently, plasma glycerophospholipid and sphingolipid species are also dysregulated in alcohol-dependent patients [16]. ...
By catalyzing the hydrolysis of sphingomyelin into ceramide, acid sphingomyelinase (ASM) changes the local composition of the plasma membrane with effects on receptor-mediated signaling. Altered enzyme activities have been noted in common human diseases, including alcohol dependence. However, the underlying mechanisms remain largely unresolved. Blood samples were collected from early-abstinent alcohol-dependent in-patients (n[♂] = 113, n[♀] = 87) and matched healthy controls (n[♂] = 133, n[♀] = 107), and analyzed for routine blood parameters and serum ASM activity. We confirmed increased secretory ASM activities in alcohol-dependent patients compared to healthy control subjects, which decreased slightly during detoxification. ASM activity correlated positively with blood alcohol concentration, withdrawal severity, biomarkers of alcohol dependence (liver enzyme activities of gamma-glutamyl transferase, alanine aminotransferase, aspartate aminotransferase; homocysteine, carbohydrate-deficient transferrin; mean corpuscular volume, and creatine kinase). ASM activity correlated negatively with leukocyte and thrombocyte counts. ASM and gamma-glutamyl transferase were also associated in healthy subjects. Most effects were similar for males and females with different strengths. We describe previously unreported associations between ASM activity and markers of liver damage and myelosuppression. Further research should investigate whether this relationship is causal, or whether these parameters are part of a common pathway in order to gain insights into underlying mechanisms and develop clinical applications.
... Activity of ASM is sensitive to cellular stress and is activated, among others, by TNF-α (22), oxidative stress (23), and ionizing radiation (24). In line with these findings, recent studies provided evidence that both stress-associated disorders, as for instance major depression (25), chronic heart failure (26), acute and chronic alcohol consumption (27,28), and chronic hepatitis C infection (29), as well as various chronic unpredictable stressors (25,30), have been associated with increased ASM activity and Cer levels, respectively. ...
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)-α (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.
... In one pathway, al- cohol addiction develops first with a depression occurring as induced by the alcohol consumption. This pathway is supported by findings showing that alcohol enhances the activity of ASM and results in in- creased ceramide levels in cell cultures ( Pascual et al., 2003;Saito et al., 2005), in rodent models ( Saito et al., 2010;Liangpunsakul et al., 2012), and in humans ( Reichel et al., 2010Reichel et al., , 2011). In another pathogenetic pathway, however, the depression manifests first leading to an en- hanced alcohol consumption and finally addiction (Room, 2000;Schuckit et al., 1997Schuckit et al., , 2006Schuckit et al., , 2007Schuckit et al., , 2013. ...
Based on their pharmacological properties, psychoactive drugs are supposed to take control of the natural reward system to finally drive compulsory drug seeking and consumption. However, psychoactive drugs are not used in an arbitrary way as pure pharmacological reinforcement would suggest, but rather in a highly specific manner depending on non-pharmacological factors. While pharmacological effects of psychoactive drugs are well studied, neurobiological mechanisms of non-pharmacological factors are less well understood. Here we review the emerging neurobiological mechanisms beyond pharmacological reinforcement which determine drug effects and use frequency. Important progress was made on the understanding of how the character of an environment and social stress determine drug self-administration. This is expanded by new evidence on how behavioral alternatives and opportunities for drug instrumentalization generate different patterns of drug choice. Emerging evidence suggests that the neurobiology of non-pharmacological factors strongly determines pharmacological and behavioral drug action and may, thus, give rise for an expanded system's approach of psychoactive drug use and addiction.
... Thus, it has been demonstrated that chronic mild stress which is used as a model to induce depression results in increases in sphingolipids in the brain (Oliveira et al. 2016;Schneider et al. 2017) and that sphingolipid ceramide and elevation of its synthesizing enzyme, acid sphingomyelinase (ASM) may directly precipitate depression (Gulbins et al. 2013). Interestingly, elevated ASM is also observed in depressed patients as well as in AUD, both of which show normalization of this enzyme following pharmacotherapy or abstinence, respectively (Kornhuber et al. 2005;Reichel et al. 2010). Normalization of ASM and depressive-like behavior may also be brought about by alcohol self-administration in a mouse model of depression, suggesting antidepressant effects of low alcohol dose mediated through sphingolipids . ...
The dose-dependent effects of alcohol, where the initial euphoric and stimulant effects initiated by the exposure to low ethanol levels can quickly lead to a deadly consequence are well established. Thus, high blood alcohol concentration (BAC), as seen in alcoholics, can cause significant damage to various organs. At low concentrations (e.g., 10 mg% or lower), however, beneficial effects of alcohol, particularly on cardiovascular function have been reported. Although, the latter assertion has been challenged by recent epidemiological studies, protective effects of low alcohol concentrations in vitro and in vivo relevant to the central nervous system (CNS) is well documented. In this review, the mechanism(s) leading to the detrimental effects of high BAC, as well as the beneficial effects of low BAC will be discussed. In addition, gender consideration is touched upon. Although further investigation is clearly warranted, it may be concluded that at least some of the beneficial outcomes of low BAC, including possible neuroprotection and antidepressant-like effects, may be due to elevation of the neurotropic factors and reduction of inflammatory mediators, whereas detrimental outcomes associated with high BAC, including neurotoxicity and depressive-like behavior may be due to reduction in neurotropic factors and elevation of inflammatory mediators. Furthermore, new research strategies are suggested.
... Altered activities or levels of their respective substrates or products have been found under pathological conditions of several common diseases [8,9]. For example, the activity of the lysosomal form of ASM (L-ASM) was found to increase in peripheral blood mononuclear cells of alcohol-dependent patients when compared to healthy controls and to decrease during withdrawal [10]. An even stronger effect on the secretory form (S-ASM) in plasma was observed in these individuals [11,12]. ...
As part of the sphingomyelin pathway, sphingomyelinases and ceramidases have attracted much attention in basic as well as clinical research. However, current assays still often rely on a radioactive substrate, extensive manual purification steps, and hazardous solvents for chromatographic analysis. We here show the equivalence of a fluorescent sphingomyelin substrate and present a new versatile solvent replacing the chloroform/methanol mixture. By further modifications including the omission of the manual extraction steps, chloroform and methanol are eliminated from the entire procedure and render the assay flexible to repeated analyses at multiple time intervals. These improvements allow for the rapid detection of both enzymes in a high throughput microtiter format. Moreover, we demonstrate the relevance of the plastic assay material and the interchangeability between serum and different plasma sources.
... Sphingolipids are involved in fundamental cellular processes, such as differentiation, senescence, and apoptosis [22], as well as in behavioral adaptations [29,53] and psychiatric disorders [51,65]. It was shown that depressed patients and alcohol dependent patients during withdrawal show increased ASM activity in peripheral blood mononucleated cells and in the plasma [36,58,59]. Transgenic mice over-expressing ASM (tgASM) display a reduced neurogenesis in the hippocampus and a depression/anxiety phenotype, which can be reversed by the functional inhibitors of ASM [21,38]. ...
Alcohol is a widely consumed drug that can lead to addiction and severe brain damage. However, alcohol is also used as self-medication for psychiatric problems, such as depression, frequently resulting in depression-alcoholism comorbidity. Here, we identify the first molecular mechanism for alcohol use with the goal to self-medicate and ameliorate the behavioral symptoms of a genetically induced innate depression. An induced over-expression of acid sphingomyelinase (ASM), as was observed in depressed patients, enhanced the consumption of alcohol in a mouse model of depression. ASM hyperactivity facilitates the establishment of the conditioned behavioral effects of alcohol, and thus drug memories. Opposite effects on drinking and alcohol reward learning were observed in animals with reduced ASM function. Importantly, free-choice alcohol drinking—but not forced alcohol exposure—reduces depression-like behavior selectively in depressed animals through the normalization of brain ASM activity. No such effects were observed in normal mice. ASM hyperactivity caused sphingolipid and subsequent monoamine transmitter hypo-activity in the brain. Free-choice alcohol drinking restores nucleus accumbens sphingolipid- and monoamine homeostasis selectively in depressed mice. A gene expression analysis suggested strong control of ASM on the expression of genes related to the regulation of pH, ion transmembrane transport, behavioral fear response, neuroprotection and neuropeptide signaling pathways. These findings suggest that the paradoxical antidepressant effects of alcohol in depressed organisms are mediated by ASM and its control of sphingolipid homeostasis. Both emerge as a new treatment target specifically for depression-induced alcoholism.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-016-1658-6) contains supplementary material, which is available to authorized users.
... Finally, plasma A-SMase levels is associated with psychological variables such as craving, withdrawal symptoms and depression [94], in line with the data revealing A-SMase as a target of antidepressant drugs such as amitriptyline and fluoxetine [95]. In alcohol-dependent patients A-SMase activity is increased in peripheral blood cells and in plasma during acute intoxication and then decreases during withdrawal [96,97]. Interestingly, the levels of A-SMase activity were demonstrated to be dependent on sex: in alcohol-dependent male patients A-SMase activity started at higher levels and decreased to lower levels during long-term detoxification when compared to female patients [94]. ...
Autophagy, the main intracellular process of cytoplasmic material degradation, is involved in cell survival and death. Autophagy is regulated at various levels and novel modulators of its function are being continuously identified. An intriguing recent observation is that among these modulators is the sphingolipid metabolising enzyme, Acid Sphingomyelinase (A-SMase), already known to play a fundamental role in apoptotic cell death participating in several pathophysiological conditions. In this review we analyse and discuss the relationship between autophagy and A-SMase describing how A-SMase may regulate it and defining, for the first time, the existence of an A-SMase-autophagy axis. The imbalance of this axis plays a role in cancer, nervous system, cardiovascular, and hepatic disorders.
... ASM genotypes and ASM activity were initially analysed in 156 healthy individuals enrolled in the GENES study [45,[48][49][50]. Further data were obtained from the ASPECT control group (n=133) [31]. All studies were carried out at the Department of Psychiatry and Psychotherapy at the University of Erlangen-Nuremberg. ...
Acid sphingomyelinase (ASM) catalyses the hydrolysis of sphingomyelin into ceramide, which acts as a lipid messenger that regulates important cellular functions. Deregulated ASM activity has been reported for different common diseases, but the mechanisms regulating ASM activity are still debated. ASM contains an exceptional signal peptide which is polymorphic due to a variable number of a hexanucleotide sequence that determines the length of the hydrophobic core. We investigated the impact of the signal peptide polymorphism on the regulation of ASM activity and secretion in vivo and in vitro.
... Liver biopsies from patients with alcoholic hepatitis exhibited 2-3-fold higher expression levels of ASM and endoplasmatic reticulum stress markers at the mRNA level than samples from control subjects [14]. We have previously observed an altered peripheral ASM activity in alcohol dependent patients: L-ASM levels were increased in acutely intoxicated individuals' blood mononuclear cells and decreased during withdrawal [15]. Similarly, the activity of plasmatic S-ASM dropped remarkably by about 50% from day 0 to day 10 of physical withdrawal to levels that were still above those of healthy controls [16]. ...
In vitro and in vivo studies have demonstrated the role of the acid sphingomyelinase (ASM) in pathophysiological processes and alterations in response to ethanol exposure. Cellular and plasmatic ASM activities are increased in male alcohol dependent patients and decrease during physical withdrawal.
Methods:
Here, we analyzed the time course of ASM in male and also female acutely intoxicated patients during alcohol withdrawal and compared the activity levels to those under long-term maintenance treatment. Craving and further psychometric parameters were assessed by questionnaires.
Results:
The gradual decrease of serum ASM was confirmed in males (p<0.001) and continued to lower activities in long-term patients (p=0.001). The trend was similar in females (p=0.178), although the initial enzyme activities were significantly lower (p=0.035). ASM activity strongly correlated with the body mass index in males. The initial ASM activity and its decline during the first two days were associated with the improvement in scores for the Beck depression inventory, the obsessive compulsive drinking and the withdrawal syndrome scales.
Conclusion:
These data support the potential of ASM as a biomarker for the course of withdrawal therapy in males and provide the first associations of this enzyme with psychological variables such as craving and depression.
... 36,[38][39][40][41] A recent in vivo study on patients with alcohol dependence reported alcohol-induced release of phosphorylcholine from sphingomyelins in the peripheral blood cells confirming alcohol-induced activation of ASM. 42 There is a direct correlation between PC concentrations and phosphatidylethanol (PEth). PEth is a clinical biomarker of the past 1-2 weeks of moderate-to-heavy alcohol consumption. ...
Alcohol consumption is one of the world's major risk factors for disease development. But underlying mechanisms by which moderate-to-heavy alcohol intake causes damage are poorly understood and biomarkers are sub-optimal. Here, we investigated metabolite concentration differences in relation to alcohol intake in 2090 individuals of the KORA F4 and replicated results in 261 KORA F3 and up to 629 females of the TwinsUK adult bioresource. Using logistic regression analysis adjusted for age, body mass index, smoking, high-density lipoproteins and triglycerides, we identified 40/18 significant metabolites in males/females with P-values <3.8E-04 (Bonferroni corrected) that differed in concentrations between moderate-to-heavy drinkers (MHD) and light drinkers (LD) in the KORA F4 study. We further identified specific profiles of the 10/5 metabolites in males/females that clearly separated LD from MHD in the KORA F4 cohort. For those metabolites, the respective area under the receiver operating characteristic curves were 0.812/0.679, respectively, thus providing moderate-to-high sensitivity and specificity for the discrimination of LD to MHD. A number of alcohol-related metabolites could be replicated in the KORA F3 and TwinsUK studies. Our data suggests that metabolomic profiles based on diacylphosphatidylcholines, lysophosphatidylcholines, ether lipids and sphingolipids form a new class of biomarkers for excess alcohol intake and have potential for future epidemiological and clinical studies.
... ASM, can be activated via engagement of the TNF-receptor super-family members—Fas (Cremesti et al., 2001; Grassme et al., 2001a,b), CD40 (Grassme et al., 2002), DR5 (Dumitru and Gulbins, 2006) and TNF (Garcia-Ruiz et al., 2003 ). Furthermore, a number of groups have demonstrated activation of ASM by various stress stimuli, such as LPS (Pfeiffer et al., 2001 ), disruption of integrin signaling (Erdreich-Epstein et al., 2005), engagement of the platelet-activating factor-receptor (Goggel et al., 2004), UV-light (UV-A (Zhang et al., 2001) and UV-C (Kashkar et al., 2005)), heat (Chung et al., 2003), alcohol (Reichel et al., 2010), oxidative stress (Sanvicens and Cotter, 2006 ), chemotherapeutic agents like cisplatin (Boitrel et al., 2011), gemcitabine (Modrak et al., 2004), doxorubicin (Morita and Tilly, 2000), or ionizing radiation (Paris et al., 2001) and accumulation of Cu 2+ (Lang et al., 2007). All these stimuli may ultimately lead to ceramide production with further consequences on plasma membrane and cell fate. ...
Recently it has become clear that exposure to xenobiotics may result in various forms of cell death; not only passive cell deaths like necrosis, or programmed cell deaths such as apoptosis, but also regulated necrosis, autophagy, senescence, or mitotic catastrophe. Complex cell signaling networks influence the processing of cell death. Furthermore, recent research has revealed early complex molecular interactions between organelles prior to the final triggering of cell death. The plasma membrane may play an important role in the early cell death signaling events. Regarding this latter aspect, drugs and environmental pollutants have been reported to affect plasma membrane characteristics which may further affect cell fate. Changes in membrane fluidity or in composition and function of specialized membrane microdomains (plasma membrane remodeling) have been proven to be involved in the regulation of many important physiological signaling pathways, including cell death. Furthermore, it has been suggested that a crosstalk between chemical-induced cellular membrane effects and other organelles may be of vital importance to explain the final outcome of chemical exposure. Here, we review the effects of plasma membrane remodeling on cell survival and cell death; we describe how the cell signaling pathways activated by changes in plasma membrane characteristics may influence cell fate. Since plasma membrane function plays an important role in the regulation of a number of cellular responses, it has been implicated in the development or progress of several diseases. A better knowledge of the effects of various chemicals on plasma membrane remodeling may be important for understanding the pathogenesis of major diseases, and may assist in developing new therapeutic strategies.
... Dramatically decreased ASM activity, due to inherited sequence variations in the SMPD1 gene coding for ASM, is the cause of the type A and B forms of Niemann-Pick disease [4]. Elevated ASM activity is associated with different neuro-psychiatric disorders like Alzheimer's dementia [5], status epilepticus [6], alcoholism [7] and major depression disorder [8]. Due to these pathological consequences, the tight regulation of ASM activity is crucial for normal cellular function. ...
Acid sphingomyelinase (ASM) hydrolyses sphingomyelin and generates the lipid messenger ceramide, which mediates a variety of stress-related cellular processes. The pathological effects of dysregulated ASM activity are evident in several human diseases and indicate an important functional role for ASM regulation. We investigated alternative splicing as a possible mechanism for regulating cellular ASM activity.
We identified three novel ASM splice variants in human cells, termed ASM-5, -6 and -7, which lack portions of the catalytic- and/or carboxy-terminal domains in comparison to full-length ASM-1. Differential expression patterns in primary blood cells indicated that ASM splicing might be subject to regulatory processes. The newly identified ASM splice variants were catalytically inactive in biochemical in vitro assays, but they decreased the relative cellular ceramide content in overexpression studies and exerted a dominant-negative effect on ASM activity in physiological cell models.
These findings indicate that alternative splicing of ASM is of functional significance for the cellular stress response, possibly representing a mechanism for maintaining constant levels of cellular ASM enzyme activity.
... Agents that reduce ASM activity and thereby also ceramide levels tend to attenuate receptor-mediated apoptosis, stress stimuli-induced apoptosis as well as growth factor-deprivation-mediated apoptosis and promote cell proliferation [47][48][49][50][51][52]. Thus, ASM-inhibitors potentially have antiapoptotic and neuroprotective effects and may therefore be used in the treatment of disorders such as brain ischemia, stroke [12,53], ethanol-induced neuronal cell death [54,55], Alzheimer's dementia [22,56,57], Parkinson's disease, Chorea Huntington, spinal cord injury [58], seizure disorder [59], glaucoma, and to protect against neurodegeneration occurring in multiple sclerosis. Furthermore, such drugs should prevent the radiation-and chemotherapy-induced lethal gastrointestinal syndrome [8], and should be helpful in the endotoxic shock syndrome [60], in severe sepsis [61] and in liver cell death and anaemia occurring in Wilson's disease [62]. ...
Acid sphingomyelinase (ASM) is an important lipid-metabolizing enzyme cleaving sphingomyelin to ceramide, mainly within lysosomes. Acid ceramidase (AC) further degrades ceramide to sphingosine which can then be phosphorylated to sphingosine-1-phosphate. Ceramide and its metabolite sphingosine-1-phosphate have been shown to antagonistically regulate apoptosis, cellular differentiation, proliferation and cell migration. Inhibitors of ASM or AC therefore hold promise for a number of new clinical therapies, e.g. for Alzheimer's disease and major depression on the one hand and cancer on the other. Inhibitors of ASM have been known for a long time. Cationic amphiphilic substances induce the detachment of ASM protein from inner lysosomal membranes with its consecutive inactivation, thereby working as functional inhibitors of ASM. We recently experimentally identified a large number of hitherto unknown functional inhibitors of ASM and determined specific physicochemical properties of such cationic amphiphilic substances that functionally inhibit ASM. We propose the acronym "FIASMA" (Functional Inhibitor of Acid SphingoMyelinAse) for members of this large group of compounds with a broad range of new clinical indications. FIASMAs differ markedly with respect to molecular structure and current clinical indication. Most of the available FIASMAs are licensed for medical use in humans, are minimally toxic and may therefore be applied for disease states associated with increased activity of ASM.
Alcohol-associated liver disease due to harmful alcohol use and non-alcoholic fatty liver disease associated with metabolic syndrome are the two most common liver diseases worldwide. Control of respective risk factors is the cornerstone in the long-term management of these diseases. Furthermore, there are no effective therapies. Both diseases are characterized by metabolic derangements, thus, the focus of this review is to broaden our understanding of metabolic targets investigated in non-alcoholic fatty liver disease, and how these can be applied to alcohol-associated liver disease. Conserved pathogenic pathways such as dysregulated lipid metabolism, cell death pathways including apoptosis, activation of innate immune cells and stellate cells mediate both alcohol and non-alcoholic fatty liver diseases, resulting in histological abnormalities of steatosis, inflammation, fibrosis, and cirrhosis. However, pathways such as gut microbiome changes, glucose metabolism and insulin resistance, inflammatory signaling, and micro-RNA abnormalities are distinct in these two diseases. In this review article, we describe conserved and distinct pathogenic pathways highlighting therapeutic targets which may be of potential in both diseases, and those which are unique to each disease.
Depression is a severe and common mental illness worldwide. According to the most recent report from the World Health Organization (WHO, January 2020), more than 264 million peo- ple of all ages suffer from depression globally. The acid sphingomyelinase (ASM)/ceramide system has been advanced as a major player in the pathogenesis of depression. Indeed, the activity of the enzyme ASM, which converts sphingomyelin into ceramide, is enhanced in de- pressed patients, with higher concentrations of plasma ceramide than in healthy individuals. Furthermore, antidepressant drugs such as fluoxetine act as functional inhibitors of ASM (FI- ASMAs). However, the precise effects of the ASM/ceramide system on neuronal excitability remain elusive. In this study, I addressed this issue by employing the specific ASM inhibitor (ARC39) and ceramides on wild type (WT), ASM knockout (koASM) and ASM overexpressing (oeASM) mice to explore the impact of the ASM/ceramide system on neuronal excitability in the hippocampus. Whole-cell patch-clamp recordings were performed on hippocampal cells (CA1 pyramidal cells and dentate gyrus granule cells) in brain slices from adult WT, koASM and oeASM mice. My data show almost no significant differences in neuronal excitability among those three genotypes. However, the specific ASM inhibitor ARC39 (1 μM) strongly reduced the excitabil- ity of neurons from ventral hippocampus of WT and oeASM mice. No such inhibitory effect of ARC39 was observed in koASM pyramidal cells, demonstrating the drug ́s specific action on ASM. The inhibitory action of ARC39 was strongly abrogated when GABAA receptors were blocked. In some neurons, I even observed excitatory effects of the drug. Intriguingly, ARC39 also affected glutamatergic transmission, as indicated by the following observations: (i) The drug enhanced both frequency and amplitude of spontaneous excitatory postsynaptic cur- rents (spEPSCs), and (ii) elevated frequency (but not amplitude) of miniature EPSCs (mEPSCs). To dissect the effects of ARC39 on the intrinsic excitability of CA1 pyramidal cells, I applied both picrotoxin and kynurenic acid to block fast GABAergic and glutamatergic synaptic inputs, respectively. In such functionally isolated neurons, I obtained both monophasic and biphasic effects of ARC39 on neuronal firing, suggesting that ARC39 might alter the balance between excitatory and inhibitory mechanisms in a cell-specific fashion. Application of C2 ceramide produced mixed effects on cell excitability along the hippocampal longitudinal axis. The C2 ceramide-induced excitation in dorsal CA1 pyramidal cells (100% ex- citation) was shifted towards mixed effects in ventral CA1 pyramidal cells (54.5% excitation), in which also inhibitory effects were seen. In dorsal granule cells, the uniformly excitatory effect of ceramide (100%) in WT mice was preserved in koASM mice (100%), suggesting that the reduced ceramide level in koASM mice fails to alter the responsiveness to C2 ceramide. Furthermore, voltage-clamp recordings from granule cells revealed that the ceramide-in- duced apparent inward currents reversed around -97 mV, close to the reversal potential of potassium channels. My data suggest that the ASM/ceramide system exerts a complex pat- tern of acute electrophysiological effects in the hippocampus, depending in part on which region along its longitudinal axis is investigated. In summary, the ASM inhibitor ARC39 reveals a tonic control of the enzyme over hippocampal excitability, which involves the intrinsic properties of CA1 pyramidal cells as well as their in- hibitory and excitatory synaptic inputs. It remains to be determined if these newly identified electrophysiological effects of ASM are altered in animal models of depression and if they represent targets of the antidepressant action of drugs like fluoxetine that act as functional inhibitors of this enzyme.
Acid sphingomyelinase (ASM) regulates a variety of physiological processes and plays an important role in emotional behavior. The role of ASM in fear-related behavior has not been investigated so far. Using transgenic mice overexpressing ASM (ASMtg) and ASM deficient mice, we studied whether ASM regulates fear learning and expression of cued and contextual fear in a classical fear conditioning paradigm, a model used to investigate specific attributes of post-traumatic stress disorder (PTSD). We show that ASM does not affect fear learning as both ASMtg and ASM deficient mice display unaltered fear conditioning when compared to wild-type littermates. However, ASM regulates the expression of contextual fear in a sex-specific manner. While ASM overexpression enhances the expression of contextual fear in both male and female mice, ASM deficiency reduces the expression of contextual fear specifically in male mice. The expression of cued fear, however, is not regulated by ASM as ASMtg and ASM deficient mice display similar tone-elicited freezing levels. This study shows that ASM modulates the expression of contextual fear but not of cued fear in a sex-specific manner and adds a novel piece of information regarding the involvement of ASM in hippocampal-dependent aversive memory.
Background
Prior research suggests that psychiatric disorders could be linked to increased mortality among patients with COVID-19. However, whether all or specific psychiatric disorders are intrinsic risk factors of death in COVID-19, or whether these associations reflect the greater prevalence of medical risk factors in people with psychiatric disorders, has yet to be evaluated.
Methods
We performed an observational multicenter retrospective cohort study to examine the association between psychiatric disorders and mortality among patients hospitalized for laboratory-confirmed COVID-19 at 36 Greater Paris University hospitals.
Results
Of 15,168 adult patients, 857 (5.7%) had an ICD-10 diagnosis of psychiatric disorder. Over a mean follow-up of 14.6 days (SD=17.9), death occurred in 326/857 (38.0%) patients with a diagnosis of psychiatric disorder versus 1,276/14,311 (8.9%) in patients without such a diagnosis (OR=6.27; 95%CI=5.40-7.28; p<0.01). When adjusting for age, sex, hospital, current smoking status, and medications according to compassionate use or as part of a clinical trial, this association remained significant (AOR=3.27; 95%CI=2.78-3.85; p<0.01). However, additional adjustments for obesity and number of medical conditions resulted in a non-significant association (AOR=1.02; 95%CI=0.84-1.23; p=0.86). Exploratory analyses following the same adjustments suggest that a diagnosis of mood disorders was significantly associated with reduced mortality, which might be explained by the use of antidepressants.
Conclusions
These findings suggest that the increased risk of COVID-19-related mortality in individuals with psychiatric disorders hospitalized for COVID-19 might be explained by the greater number of medical conditions and the higher prevalence of obesity in this population, but not by the underlying psychiatric disease.
Hintergrund und Ziele. Der schädliche und unangemessene Konsum von Alkohol ist ein weltweites Problem und gewinnt in der heutigen Zeit und in den westlichen Industrieländern zunehmend an Bedeutung. Er kann neben einer Alkoholkonsumstörung und Alkoholsucht zu vielen somatischen Erkrankungen, wie zum Beispiel Erkrankungen des Herz-Kreislaufsystems oder Tumorerkrankungen führen. Schließlich können auch psychiatrische Erkrankungen wie die Depression und Angststörungen auftreten oder sich als Folge dieser entwickeln. Ein Erklärungsansatz für diese Komorbiditäten liegt in der Regulation des Sphingolipidhaushalts durch das Enzym Neutrale Sphingomyelinase 2 (NSM). In der vorliegenden Arbeit wurde im pharmakologischen Modell der NSM-Inhibition durch GW4869 das Alkoholtrinkverhalten von Mäusen sowie deren Emotionalität untersucht und im Hinblick auf die Regulation der Sphingolipidhomöostase durch die NSM analysiert. Die Hypothese lautete in diesem Zusammenhang, dass der NSM-Inhibitor GW4869 das Trinkverhalten und das emotionale Verhalten in entsprechenden Tests verändert und diese Effekte geschlechtsabhängig sind. Methoden. Für die Experimente wurden 31 naive C57Bl6-Mäuse verwendet, jeweils 16 Männchen und 15 Weibchen. Grundvoraussetzungen für die Durchführung der Experimente war es, der Hälfte der Tiere (sowohl Männchen als auch Weibchen) den NSM-Inhibitor GW4869 kontinuierlich mit einer Dosis von 2 mg/kg/Tag mittels osmotischer Minipumpen zu verabreichen, um so die Effekte der pharmakologischen Herunterregulierung der NSM-Aktivität auf Trinkverhalten und Emotionalität zu untersuchen. Der Kontrollgruppe wurde Saline (0,9 % NaCl) verabreicht (n = 8 bzw. 7 Mäuse/Versuchsgruppe). Für die Alkoholtrinkstudie wurde ein Zwei-Flaschen-Wasser-Alkohol-Wahlparadigma benutzt und für die Verhaltensexperimente dienten der Open Field Test (OFT), das Elevated Plus Maze (EPM) und das Novelty Suppressed Feeding (NSF). Ergebnisse. In der hier durchgeführten Alkoholtrinkstudie konnte herausgefunden werden, dass die pharmakologische Hemmung der NSM durch GW4869 bei Mäusen zu einem veränderten Trinkverhalten führt und dieser Effekt geschlechtsabhängig ist. Es zeigten sowohl GW4869-Weibchen als auch GW4869-Männchen einen höheren Alkoholkonsum gegenüber der mit Saline behandelten Kontrollgruppe. Insbesondere bei den GW4869-Weibchen gab es mit dem starken Anstieg des Alkoholkonsums und dem gleichzeitig starken Abfall im Wasserkonsums eine klare Verschiebung der Präferenz hin zum Alkohol bei hohen Alkoholkonzentrationen (12 und 16 Vol.-%). Bei den GW4869-Männchen lag die Alkoholpräferenz gegenüber Wasser bei den mittleren Alkoholkonzentrationen am höchsten. In der hier durchgeführten experimentellen Verhaltensstudie konnte außerdem beobachtet werden, dass die pharmakologische Hemmung der NSM durch GW4869 zu einem sichtbaren Effekt auf das Angst- und Depressionsverhalten führt und auch dieser geschlechtsabhängig ist. Der depressive Verhaltenseffekt war ausschließlich bedingt durch die Männchen mit einer signifikant höheren Latenzzeit bis zum ersten Fressen im NSF im Vergleich zur Kontrollgruppe. Hinsichtlich Angstverhalten zeigte sich insgesamt die Tendenz zum anxiogenen Effekt bei den GW4869-Weibchen im OFT. Schlussfolgerung. Insgesamt konnte mit den Ergebnissen meiner Arbeit bestätigt werden, dass die NSM bei neuronalen und psychiatrischen Pathologien eine bedeutende Funktion einnimmt. Die NSM scheint nach Zusammenschau der oben genannten Ergebnisse Einfluss auf die Regulation des Trink- und des Affekt-verhaltens zu nehmen. Eine Blockade der NSM durch GW4869 und damit eine Verringerung der NSM-Aktivität erhöht demnach das Konsumrisiko und die Konsummenge von Alkohol, insbesondere bei Frauen. Das wiederum bedeutet, dass der NSM-Inhibitor GW4869 ein essenzieller Modulator des Alkoholkonsums ist. Dieser Effekt ist stark weiblich dominiert und damit geschlechtsabhängig. Die NSM-Hemmung scheint geringere und dennoch sichtbare Einflüsse auf das Affekt-verhalten, in Form von depressiven Effekten bei Männern und anxiogenen Effekten bei Weibchen, zu haben. Insgesamt leistet diese Arbeit einen Beitrag zum besseren Verständnis der Regulationsmechanismen durch die NSM, mit dem Ziel psychiatrische Erkrankungen wie die Alkoholsucht, Depression und Angststörungen in Zukunft durch neue Behandlungsstrategien noch besser therapieren zu können.
Hintergrund und Ziele In der westlichen Welt wird Alkohol von der Mehrheit der Erwachsenen regelmäßig konsu- miert, trotz eines nicht unerheblichen Risikos für gesundheitliche Folgen, wie psychische und physische Schäden. Neben den medizinischen Schäden durch Alkohol, kommt es auch zu wirt- schaftlichen Folgen und schwerwiegenden sozialen und familiären Problemen. Der volkswirt- schaftliche Schaden durch Alkohol beläuft sich auf rund 40 Millionen Euro pro Jahr. So konsumieren circa 6,7 Millionen Menschen alleine in Deutschland Alkohol in einer ge- sundheitlich riskanten Form und 1,6 Millionen Menschen im Alter von 18-64 Jahren gelten in Deutschland als alkoholabhängig. In diesem Zusammenhang ist der sehr unkritische Um- gang und die gesellschaftliche Akzeptanz des regelmäßigen Konsums von Alkohol als äußerst problematisch anzusehen. Diese Situation birgt eine weitere Erschwerung in der Therapie der Erkrankung. Es gibt keine einfache spezifische Therapie, vielmehr ist die Behandlung der Alko- holsucht sehr komplex, erfordert die volle Bereitschaft des Betroffenen und seiner Angehörigen und dauert im Grunde ein Leben lang. Aufgrund der mangelnden Behandlungsoptionen ist die Suche nach weiteren therapeutischen Möglichkeiten Gegenstand der aktuellen Forschung. In den Fokus der Forschung sind hier die Sphingolipide gerückt, die im Gehirnstoffwechsel ei- ne wichtige, aber zum Teil noch unverstandene Rolle übernehmen. Als Enzym der Familie der Sphingomyelinasen (SMasen) ist auch die neutrale Sphingomyelinase (NSM) in den Hirnstoff- wechsel eingebunden. Das Ziel dieser Arbeit war herauszufinden, welche Auswirkungen der heterozygote Knockout der NSM in Kombination mit dem Konsum von Alkohol einerseits auf die Entwicklung von Sucht und anderseits auf Morphologie von Gehirn und Knochen hat. Material und Methoden Über einen Zeitraum von 43 Tagen wurde das Trinkverhalten von weiblichen fragilitas ossium (fro)-Mäusen mit einem heterozygoten Defekt der NSM sowie wildtyp (wt)-Mäusen als Kon- trollgruppe beobachtet. In der Versuchsgruppe wurde sowohl den fro- als auch den wt-Mäusen der freiwillige Konsum einer 16 vol.%-Ethanollösung angeboten. Der Kontrollgruppe hingegen stand ausschließlich Wasser zur Verfügung. Das Trinkverhalten der Mäuse wurde analysiert, um so eine Aussage zu Konsum und Präferenz zu treffen. Zur Darstellung von Effekten des Alkoholkonsums, der Defizienz der NSM und des Zusammenspiels beider Faktoren auf Hirn- und Knochengewebe, schloss sich an die Phase des Trinkens bei allen 31 Mäusen eine Messung des Gehirns mittels Magnetresonanztomographie (MRT) und eine Computertomographie (CT)- gestützte Messung des kompletten Skeletts an. Im Rahmen der Auswertung der Messungen mit dem Programm „aycan OSiriX mit Segmenta- tion“ wurden die Werte für Hirnvolumina und Knochendichte der fro-Mäuse mit den jeweiligen Kontrollgruppen verglichen. Ergebnisse und Beobachtungen Mäuse mit einem heterozygoten Defekt der NSM zeigten eine erhöhte Präferenz für Alkohol und einen erhöhten Konsum von Alkohol im Vergleich zu wt-Mäusen. Der totale Flüssigkeits- konsum und der Konsum von Leitungswasser lagen unter dem Wert der wt-Mäuse. Bei den Auswertungen der Knochendichte durch eine CT-Untersuchung zeigten sich keine signifikanten Unterschiede in der Knochendichte zwischen wt-Mäusen und fro-Mäusen, unabhängig davon, ob die Mäuse Alkohol oder Leitungswasser tranken. In den Messungen der Hirnvolumina zeig- ten sich jedoch deutliche Unterschiede zwischen fro-Mäusen, die ausschließlich Wasser tranken und derer, die freiwillig Ethanol konsumieren konnten. Bei Ethanol-konsumierenden heterozy- goten fro-Mäusen kam es zu einer Reduktion der Volumina in gemessenen Hirnregionen. Diese Veränderung war vor allem in den Bereichen des dorsalen Hippocampus signifikant und im Be- reich der Inselrinde trendsignifikant. Bei wt-Mäusen konnte diese Reduktion trotz den Konsums von Ethanol nicht aufgezeigt werden. Schlussfolgerung Der alleinige Konsum von Alkohol zeigte keinen Effekt auf die Morphologie von Strukturen im Gehirn. Obwohl Ethanol durch eine erhöhte Produktion von Ceramid eine neurotoxische Zelldegeneration auslöst, war weder die Dauer noch die konsumierte Alkoholmenge ausrei- chend, um in diesem Setting einen Effekt zu bewirken. In Interaktion mit einem heterozygoten Knockout der NSM kam es jedoch bei Konsum von Ethanol zu signifikanten Auswirkungen. Interessanterweise war das Volumen des dorsalen Hippocampus zu Beginn des Experimentes im Vergleich zur wt-Gruppe signifikant erhöht. Somit kann angenommen werden, dass die fro- Mäuse vor Beginn des Alkoholkonsums über eine erhöhte Resilienz gegen Stressoren verfügten. Trotz der eigentlich verminderten Produktion von Ceramid durch die Defizienz der NSM, traten bei gleichzeitigem Konsum von Ethanol neurotoxische Effekte und eine Verminderung defi- nierter Strukturen auf. Als signifikant ist diese Reduktion im dorsalen Hippocampus und als trendsignifikant in der Inselrinde zu werten. Dabei handelt es sich um Strukturen, die immense Wichtigkeit in der Pathogenese psychiatrischer Erkrankungen, wie Sucht und Depression, auf- weisen. Ebenfalls konnte in NSM-Knockout-Mäusen eine erhöhte Präferenz und ein erhöhter Konsum von Ethanol per se festgestellt werden. Ein Einfluss auf die Mineralisierung und Dich- te der Knochen bei fro-Mäusen durch die Mutation selbst, den Konsum von Ethanol oder das Zusammenwirken beider Faktoren war in diesem Experiment nicht festzustellen. Die Ergebnis- se belegen spezifische Auswirkungen durch das Zusammenspiel der heterozygoten Defizienz der NSM mit Ethanol auf die Entwicklung und Morphologie von Hirnstrukturen.
Despite the advancements in modern medicine, there are still difficulties in diagnosing common illnesses. The invasiveness and price of the tests used to follow up certain diseases can be a barrier to proper patient follow-up. Sphingolipids are a diverse category of lipids. They are structural molecules in cell membranes and signaling molecules involved in the regulation of crucial cell functions, including cell growth, differentiation, proliferation and apoptosis. Recent research has shown that abnormal sphingolipid metabolism is associated with genetic and metabolic disease processes. Given their crucial role to maintain homeostasis within the body, sphingolipids have been investigated as potential biomarkers to predict disease in the population. Here we discuss how sphingolipids levels are altered in different diseases, thus illustrating their possible use as diagnostic and prognostic biomarkers for disease.
Alcohol use disorder and depression show a high comorbidity at clinical level with no treatment available selectively for this condition. A hyperfunction of acid sphingomyelinase (ASM) and resulting ceramide overload were recently identified as one pathway into this comorbidity. Here we analyzed the involvement of ASM, one of the main enzymes involved in ceramide synthesis, in the molecular control of monoaminergic systems in their basal activity and in response to pharmacological and natural reinforcers. The effects of alcohol and a palatable food on the extracellular levels of dopamine (DA), serotonin (5-HT), and noradrenaline (NE) were measured by in-vivo microdialysis in ASM overexpressing mice (tgASM). We found reduced basal extracellular DA levels in the nucleus accumbens (Nac) and dorsal hippocampus (DH) of tgASM mice with little effect on 5-HT and NE levels. In contrast, ASM overexpression potentiated the DA response to alcohol (2 g/kg, i.p.) in the DH and Nac, but reduced NE responses. DA and NE responses to a food stimulus were not altered in tgASM mice, but the Nac 5-HT response was enhanced.
An immunohistochemical analysis of the DH showed a preserved dopaminergic and serotonergic innervation in tgASM mice and in mice that consumed alcohol for one month. These findings suggest a direct modulation of monoaminergic basal activity and/or responses to reinforcing stimuli by the sphingolipid regulatory enzyme ASM in mice.
Psychoactive drug use is a common behavior in many societies worldwide, frequently associated with drug instrumentalization. Regular use may develop into drug addiction, which is a severe psychiatric disorder with multiple pathological effects to virtually all organ systems. Treatment strategies for addiction are often insufficient with no broadly working pharmaco-treatment available. Recently, lipids, and particularly sphingolipids, have been considered as new mediators in the pathogenic pathways and as possible therapeutic targets for the treatment of addictive states. In our review, we discuss the contribution of sphingolipids in the development of addictive states including alcohol consumption, nicotine, amphetamine, morphine, and cocaine dependencies. Recent data show that the involvement of various classes of sphingolipids, such as sphingomyelins, ceramides, globosides, sulfatides, and cerebrosides, might explain the development of some specific features of addictive states, for example, apoptotic neurodegeneration induced by psychoactive substances. On the other hand, protective effects of sphingolipids are discussed. Sphingolipids might be a key mechanism in the development of beneficial effects of moderate alcohol consumption. Therefore, sphingolipid systems emerge as possible new pathways involved in the development of addiction and its pathophysiological consequences. However, further analysis is still needed to investigate the exact mechanisms of sphingolipid contribution and possibility of using of sphingolipids as new therapeutic targets.
Major depression is a systems disorder which impairs not only central nervous system aspects of mood and behavior but also peripheral organ systems. Current views on the pathogenesis and treatment of depression are predominantly based on proteins and transmitters and thus are difficult to reconcile central with peripheral pathomechanisms. Recent research showed that there is also a lipid-based pathway involved in the pathology of depression, which is activated by psychosocial stress, oxidative stress, or inflammation. Inducible dysfunction of the ceramide pathway, which is abundant in the brain as well as in peripheral organs, may account for mood disorder, behavioral symptoms, and further promote inflammation and oxidative stress in peripheral systems. As such, the lipid ceramide pathway may provide the missing link between brain dysfunction and somatic symptoms of depression. Pharmacological interventions that reduce ceramide abundance also show antidepressant action and may promise a better treatment of major depression.
Sphingolipids are not only structural components of biological membranes, but also play an important role in cellular signalling and, thus, are involved in cell proliferation and differentiation but also stress and cell death. It is therefore of great clinical relevance to define inhibitors of the enzymes involved in sphingolipid metabolism. Here, we describe the state of the art of functional inhibitors of the acid sphingomyelinase. The acid sphingomyelinase converts sphingomyelin to ceramide, a compound often involved in cell stress. We describe the structural and physicochemical properties, the distribution, the pharmacokinetics, the pharmocodynamics and the clinical use of direct and functional inhibitors of the acid sphingomyelinase.
Acid sphingomyelinase (ASM, EC 3.1.4.12) hydrolyzes sphingomyelin to ceramide and represents a major regulator of sphingolipid metabolism. Increased activity of ASM has been observed in a variety of human diseases, and a critical contribution of ASM to medical conditions was demonstrated in several mouse models. In agreement with increased ASM activity in cell lines treated with ethanol, we have recently found higher levels of ASM activity in peripheral blood cells of active drinkers. However, the influence of ethanol on secretory ASM (S-ASM) has not been investigated so far.
ASM activity and routine blood parameters were determined in plasma samples of 27 patients with alcohol dependence during physical withdrawal and compared to a group of 36 healthy volunteers.
Compared to the control group, patients with alcohol dependence had S-ASM activity increased by about 3-fold (141 ± 69 vs. 428 ± 220 pmol/ml/h; p < 0.001) at the beginning of physical withdrawal. During withdrawal, S-ASM activity decreased by about 50% (p < 0.001; day 0 vs. day 7 to 10) and finally approximated nearly normal values. On the day of admission, S-ASM activity correlated positively with levels of carbohydrate-deficient transferrin (r = 0.410, p = 0.034) and high-density lipoprotein cholesterol (r = 0.440, p = 0.022) and inversely with body mass index (r = -0.509; p = 0.007), glucose (r = -0.480; p = 0.011), triglycerides (r = -0.592; p = 0.001), and large unstained cells (r = -0.526; p = 0.017).
Activity of S-ASM is increased in alcohol-dependent patients and correlates with established biomarkers of excessive drinking. The increased S-ASM activity is implicated in alcohol-induced lipid alterations and might be relevant for the occurrence of alcohol-related disorders.
Alcoholism is a chronic relapsing disorder. Major characteristics observed in alcoholics during an initial period of alcohol abstinence are altered physiological functions and a negative emotional state. Evidence suggests that a persistent, cumulative adaptation involving a kindling/allostasis-like process occurs during the course of repeated chronic alcohol exposures that is critical for the negative symptoms observed during alcohol withdrawal. Basic studies have provided evidence for specific neurotransmitters within identified brain sites being responsible for the negative emotion induced by the persistent cumulative adaptation following intermittent-alcohol exposures. After an extended period of abstinence, the cumulative alcohol adaptation increases susceptibility to stress- and alcohol cue-induced negative symptoms and alcohol seeking, both of which can facilitate excessive ingestion of alcohol. In the alcoholic, stressful imagery and alcohol cues alter physiological responses, enhance negative emotion, and induce craving. Brain fMRI imaging following stress and alcohol cues has documented neural changes in specific brain regions of alcoholics not observed in social drinkers. Such altered activity in brain of abstinent alcoholics to stress and alcohol cues is consistent with a continuing ethanol adaptation being responsible. Therapies in alcoholics found to block responses to stress and alcohol cues would presumably be potential treatments by which susceptibility for continued alcohol abuse can be reduced. By continuing to define the neurobiological basis of the sustained alcohol adaptation critical for the increased susceptibility of alcoholics to stress and alcohol cues that facilitate craving, a new era is expected to evolve in which the high rate of relapse in alcoholism is minimized.
Major depression is a severe mood disorder with a lifetime prevalence of more than 10%. The pharmacokinetic hypothesis claims that a slow accumulation of antidepressant drugs by acid trapping mainly into lysosomes is responsible for the therapeutic latency and that a lysosomal target mediates the antidepressant effects. The lysosomal lipid metabolizing enzyme acid sphingomyelinase (ASM) cleaves sphingomyelin into ceramide and phosphorylcholine. In a pilot study, the activity of this enzyme was increased in peripheral blood cells of patients with major depressive disorder (MDD), making the ASM an interesting molecular target of antidepressant drugs. Indeed, several antidepressant drugs functionally inhibit ASM. The ASM/ceramide pathway might be a missing link unifying independent findings in neurobiology and the treatment of MDD such as therapeutic latency, oxidative stress, immune activation and increased risk of cardiovascular disease.
Acid sphingomyelinase (ASM) is reported to have an essential function in stress-induced apoptosis although the physiological function of ASM in receptor-triggered apoptosis is unknown. Here, we delineate a pivotal role for ASM in CD95-triggered apoptosis of peripheral lymphocytes or hepatocytes in vivo.We employed intravenous injection of anti-CD4 antibodies or phytohemagglutinin that was previously shown to result in apoptosis of peripheral blood lymphocytes or hepatocytes via the endogenous CD95/CD95 ligand system. Our results demonstrate a high susceptibility in normal mice whereas ASM knock-out mice fail to immunodeplete T cells or develop autoimmune-like hepatitis. Likewise, ASM-deficient mice or hepatocytes and splenocytes ex vivo manifest resistance to anti-CD95 treatment. These results provide in vivo evidence for an important physiological function of ASM in CD95-induced apoptosis.
We recently reported that a mild heat shock induces a long lasting stimulation of TRAIL-induced apoptosis of leukemic T-lymphocytes and myeloid cell lines, but not normal T-lymphocytes, which correlates with an enhanced ability of TRAIL to recognize its receptors. As shown here, this phenomenon could be inhibited by the xanthogenate agent D609, a sphingomyelin/ceramide pathway inhibitor. A caspase-dependent and D609-sensitive twofold increase in ceramide level was elicited by heat shock plus TRAIL combined treatment. One day after heat shock, a similar increase in ceramide was induced by TRAIL. Sphingolipids/ceramides are known to regulate membrane integrity, and heat shock increases membrane fluidity. In this regard, the heat shock plus TRAIL combined treatment resulted in a D609-sensitive membrane fluidization which was far more intense than that induced by heat shock only. We also report that membrane flui-dizers, that mimic the effect of heat shock, such benzyl alcohol and ethanol, potently stimulated TRAIL-induced apoptosis. As heat shock, these alcohols increased, in a D609-sensitive manner, membrane fluidity in the presence of TRAIL, the recognition of TRAIL death receptors, and ceramide levels. These results suggest that stress agents that trigger ceramide production and an overall increase in membrane fluidity are stimulators of TRAIL apoptosis.
T cell receptor recognition of antigen can lead either to T lymphocyte differentiation and proliferation or to a state of unresponsiveness, which is dependent on whether appropriate costimulatory signals are provided to the mature T cell. We have investigated a novel intracellular signaling pathway provided by the costimulatory molecule CD28. CD28 engagement triggers the activation of an acidic sphingomyelinase (A-SMase), which results in the generation of ceramide, an important lipid messenger intermediate. A-SMase activation by CD28 occurred in resting as well as in activated primary T cells or leukemic Jurkat cells. In contrast, ligation of either CD3 or CD2 did not result in A-SMase activation. Overexpression of recombinant A-SMase in Jurkat T cells substituted for CD28 with regard to nuclear factor-kB activation. These data suggest that CD28 provides an important costimulatory signal by activation of an acidic sphingomyelinase pathway.
Mammalian sphingomyelinases have been implicated in many important physiological and pathophysiological processes. Although several mammalian sphingomyelinases have been identified and studied, one of these, an acidic Zn2+-stimulated sphingomyelinase (Zn-SMase) originally found in fetal bovine serum, has received little attention since its first and only report 7 years ago. We now show that Zn-SMase activity is secreted by human and murine macrophages, human skin fibroblasts, microglial cells, and several other cells in culture and is markedly up-regulated during differentiation of human monocytes to macrophages. Remarkably, peritoneal macrophages from mice in which the acid SMase gene had been disrupted by homologous recombination secreted no Zn-SMase activity, indicating that this enzyme and the intracellular lysosomal SMase, which is Zn-independent, arise from the same gene. Furthermore, skin fibroblasts from patients with types A and B Niemann-Pick disease, which are known to lack lysosomal SMase activity, also lack Zn-SMase activity in their conditioned media. Chinese hamster ovary cells stably transfected with a cDNA encoding lysosomal SMase massively overexpress both cellular lysosomal SMase and secreted Zn-SMase activities. Thus, Zn-SMase arises independently of alternative splicing, suggesting a post-translational process. In summary, a wide variety of cell types secrete Zn-SMase activity, which arises from the same gene as lysosomal SMase. This secreted enzyme may play roles in physiological and pathophysiological processes involving extracellular sphingomyelin hydrolysis.
Cells from the human monocytic leukemia cell line THP-1 differentiate towards a macrophage-like phenotype when stimulated with phorbol 12-myristate -13- acetate (PMA), 1,25-dihydroxy-vitamin D3, and various other agents. We demonstrate here that the expression of the lysosomal enzyme acid sphingomyelinase (ASM; E.C. 3.1.4.12) is induced during this process and is strongly elevated in differentiated THP-1 cells, as well as in differentiated human mononuclear phagocytes. Using Northern blotting, RNase protection assay, and nuclear run-on analyses, we show that the up-regulation of ASM expression is regulated mainly at the level of transcription and that new protein synthesis is required for enhanced ASM activity. This cell-type specific induction by PMA treatment was further investigated with respect to transcriptional control. A series of 5' deletion derivatives of the upstream regulatory region were used in transient transfection assays to identify promoter elements required for basal and inducible gene expression. A PMA responsive element was localized to a region between -319 and -219 bp upstream of the initiation codon and co-transfections with transcription factor expression plasmids for AP-2 and Sp1 resulted in augmented ASM promoter activity, which was abolished when the binding sites for these two factors were deleted. Using electrophoretic mobility shift assays and supershift assays we demonstrate that this region is specifically bound by Sp1 and AP-2. These factors are present in nuclear extracts prepared from both induced and uninduced THP-1 cells. However, the intensity of the complex formed appeared to increase when nuclear extracts from PMA-treated cells were used. From these studies, we conclude that a concerted action of the transcription factors AP-2 and Sp1 is essential for the up -regulation of ASM expression during the process of macrophage differentiation.
Acid sphingomyelinase (ASM) is reported to have an essential function in stress-induced apoptosis although the physiological function of ASM in receptor-triggered apoptosis is unknown. Here, we delineate a pivotal role for ASM in CD95-triggered apoptosis of peripheral lymphocytes or hepatocytes in vivo. We employed intravenous injection of anti-CD4 antibodies or phytohemagglutinin that was previously shown to result in apoptosis of peripheral blood lymphocytes or hepatocytes via the endogenous CD95/CD95 ligand system. Our results demonstrate a high susceptibility in normal mice whereas ASM knock-out mice fail to immunodeplete T cells or develop autoimmune-like hepatitis. Likewise, ASM-deficient mice or hepatocytes and splenocytes ex vivo manifest resistance to anti-CD95 treatment. These results provide in vivo evidence for an important physiological function of ASM in CD95-induced apoptosis.
The mechanism underlying ethanol-induced apoptosis in liver cells is not clear. Sphingomyelin (SM) metabolism is a novel signal transduction pathway that has an impact on apoptosis in many cell types. We investigated whether the SM pathway is involved in ethanol-induced apoptosis in the liver. Hep G2 cells were treated with ethanol followed by assaying apoptosis, sphingomyelinase (SMase) activity, caspase-3 activity, and the changes of SM content in the cells. We found that ethanol dose-dependently increased apoptosis and the effect was accompanied by increases of caspase-3 activity and neutral SMase activity. At concentrations of 80 and 160 mM, ethanol significantly increased caspase-3 activity by 120% and neutral SMase activity by 24%. The activity of acid SMase was only slightly increased without statistical significance. C(2)-ceramide, the exogenous SM metabolite, mimicked the effects of ethanol on apoptosis and caspase-3 activation. When the SM content was determined 24 h after treatment with ethanol, its level was 15% lower than that of controls. The results indicate that metabolism of SM triggered by neutral SMase participates in ethanol-induced apoptosis in Hep G2 cells and activation of caspase-3 is involved in the apoptotic pathway.
Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.
Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.
This study addressed the contribution of acidic sphingomyelinase (ASMase) in TNF-alpha-mediated hepatocellular apoptosis. Cultured hepatocytes depleted of mitochondrial glutathione (mGSH) became sensitive to TNF-alpha, undergoing a time-dependent apoptotic cell death preceded by mitochondrial membrane depolarization, cytochrome c release, and caspase activation. Cyclosporin A treatment rescued mGSH-depleted hepatocytes from TNF-alpha-induced cell death. In contrast, mGSH-depleted hepatocytes deficient in ASMase were resistant to TNF-alpha-mediated cell death but sensitive to exogenous ASMase. Furthermore, although in vivo administration of TNF-alpha or LPS to galactosamine-pretreated ASMase(+/+) mice caused liver damage, ASMase(-/-) mice exhibited minimal hepatocellular injury. To analyze the requirement of ASMase, we assessed the effect of glucosylceramide synthetase inhibition on TNF-alpha-mediated apoptosis. This approach, which blunted glycosphingolipid generation by TNF-alpha, protected mGSH-depleted ASMase(+/+) hepatocytes from TNF-alpha despite enhancement of TNF-alpha-stimulated ceramide formation. To further test the involvement of glycosphingolipids, we focused on ganglioside GD3 (GD3) because of its emerging role in apoptosis through interaction with mitochondria. Analysis of the cellular redistribution of GD3 by laser scanning confocal microscopy revealed the targeting of GD3 to mitochondria in ASMase(+/+) but not in ASMase(-/-) hepatocytes. However, treatment of ASMase(-/-) hepatocytes with exogenous ASMase induced the colocalization of GD3 and mitochondria. Thus, ASMase contributes to TNF-alpha-induced hepatocellular apoptosis by promoting the mitochondrial targeting of glycosphingolipids.
We showed previously that alcohol exposure during in vivo brain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol-induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose-dependent manner, and that C2-ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress-related kinases, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol-induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2-ceramide. Furthermore, we show that ethanol-induced ERK activation triggers the stimulation of cyclo-oxygenase-2 (COX-2) and the release of prostaglandin E2, and that blockade of the mitogen-activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up-regulation of COX-2 induced by ethanol plus ceramide, and decreases the ethanol-induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase-ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol-induced astroglial cell death during brain development.
Acid sphingomyelinase (A-SMase) and its reaction product ceramide may play a role in the pathophysiology of depressive disorders and in the therapeutic action of antidepressive drugs. In a prospective case-control study, A-SMase activity was measured in peripheral blood mononuclear cells of 17 patients with a major depressive episode who were free of antidepressant drug therapy for at least 10 days and 8 healthy volunteers. In the patient group, A-SMase activity was correlated to the score (n=17, r=0.64, P=0.005). The patient group exhibited higher A-SMase activity compared to healthy volunteers (T=2.09, df=21.33, P<0.05). In addition, we demonstrate that the antidepressants imipramine and amitriptyline induce a long-term reduction of the activity of A-SMase in cultured cells.
NK and NKT cells play a major role in both innate immunity and in influencing the development of adaptive immune responses. CD161 (human NKR-P1A), a protein encoded in the NK gene complex, is a major phenotypic marker of both these cell types and is thought to be involved in the regulation of NK and NKT cell function. However, the mechanisms of action and signaling pathways of CD161 are poorly understood. To identify molecules able to interact with the cytoplasmic tail of human CD161 (NKR-P1A), we have conducted a yeast two-hybrid screen and identified acid sphingomyelinase as a novel intracellular signaling pathway linked to CD161. mAb-mediated cross-linking of CD161, in both transfectants and primary human NK cells, triggers the activation of acid, but not neutral sphingomyelinase. The sphingomyelinases represent the catabolic pathway for N-acyl-sphingosine (ceramide) generation, an emerging second messenger with key roles in the induction of apoptosis, proliferation, and differentiation. These data therefore define a novel signal transduction pathway for the CD161 (NKR-P1A) receptor and provide fresh insights into NK and NKT cell biology.
Although important for cellular stress signaling pathways, the molecular mechanisms of acid sphingomyelinase (ASMase) activation remain poorly understood. Previous studies showed that treatment of MCF-7 mammary carcinoma cells with the potent protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), induces a transient drop in sphingomyelin concomitant with an increase in cellular ceramide levels (Becker, K. P., Kitatani, K., Idkowiak-Baldys, J., Bielawski, J., and Hannun, Y. A. (2005) J. Biol. Chem. 280, 2606-2612). Here we show that PMA selectively activates ASMase and that ASMase accounts for the majority of PMA-induced ceramide. Pharmacologic inhibition and RNA interference experiments indicated that the novel PKC, PKCdelta, is required for ASMase activation. Immunoprecipitation experiments revealed the formation of a novel PKCdelta-ASMase complex after PMA stimulation, and PKCdelta was able to phosphorylate ASMase in vitro and in cells. Using site-directed mutagenesis, we identify serine 508 as the key residue phosphorylated in response to PMA. Phosphorylation of Ser(508) proved to be an indispensable step for ASMase activation and membrane translocation in response to PMA. The relevance of the proposed mechanism of ASMase regulation is further validated in a model of UV radiation. UV radiation also induced phosphorylation of ASMase at serine 508. Moreover, when transiently overexpressed, ASMase(S508A) blocked the ceramide formation after PMA treatment, suggesting a dominant negative function for this mutant. Taken together, these results establish a novel direct biochemical mechanism for ASMase activation in which PKCdelta serves as a key upstream kinase.
We recently reported that a mild heat shock induces a long lasting stimulation of TRAIL-induced apoptosis of leukemic T-lymphocytes and myeloid cell lines, but not normal T-lymphocytes, which correlates with an enhanced ability of TRAIL to recognize its receptors. As shown here, this phenomenon could be inhibited by the xanthogenate agent D609, a sphingomyelin/ceramide pathway inhibitor. A caspase-dependent and D609-sensitive two-fold increase in ceramide level was elicited by heat shock plus TRAIL combined treatment. One day after heat shock, a similar increase in ceramide was induced by TRAIL. Sphingolipids/ceramides are known to regulate membrane integrity, and heat shock increases membrane fluidity. In this regard, the heat shock plus TRAIL combined treatment resulted in a D609-sensitive membrane fluidization which was far more intense than that induced by heat shock only. We also report that membrane fluidizers, that mimic the effect of heat shock, such benzyl alcohol and ethanol, potently stimulated TRAIL-induced apoptosis. As heat shock, these alcohols increased, in a D609-sensitive manner, membrane fluidity in the presence of TRAIL, the recognition of TRAIL death receptors, and ceramide levels. These results suggest that stress agents that trigger ceramide production and an overall increase in membrane fluidity are stimulators of TRAIL apoptosis.
It has become increasingly difficult to find an area of cell biology in which lipids do not have important, if not key, roles as signalling and regulatory molecules. The rapidly expanding field of bioactive lipids is exemplified by many sphingolipids, such as ceramide, sphingosine, sphingosine-1-phosphate (S1P), ceramide-1-phosphate and lyso-sphingomyelin, which have roles in the regulation of cell growth, death, senescence, adhesion, migration, inflammation, angiogenesis and intracellular trafficking. Deciphering the mechanisms of these varied cell functions necessitates an understanding of the complex pathways of sphingolipid metabolism and the mechanisms that regulate lipid generation and lipid action.
T cell receptor recognition of antigen can lead either to T lymphocyte differentiation and proliferation or to a state of unresponsiveness, which is dependent on whether appropriate costimulatory signals are provided to the mature T cell. We have investigated a novel intracellular signaling pathway provided by the costimulatory molecule CD28. CD28 engagement triggers the activation of an acidic sphingomyelinase (A-SMase), which results in the generation of ceramide, an important lipid messenger intermediate. A-SMase activation by CD28 occurred in resting as well as in activated primary T cells or leukemic Jurkat cells. In contrast, ligation of either CD3 or CD2 did not result in A-SMase activation. Overexpression of recombinant A-SMase in Jurkat T cells substituted for CD28 with regard to nuclear factor-kB activation. These data suggest that CD28 provides an important costimulatory signal by activation of an acidic sphingomyelinase pathway.
We showed previously that alcohol exposure during in vivo brain development induced astroglial damage and caused cell death. Because ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, we now investigate whether ethanol-induced cell death in astrocytes is mediated by ceramide signalling pathways triggering apoptosis. Here we show that both ethanol and ceramide are able to induce apoptotic death in cultured astrocytes, in a dose-dependent manner, and that C2-ceramide addition potentiates the apoptotic effects of ethanol. Cell death induced by ethanol is associated with stimulation of neutral and acidic sphingomyelinase (SMase) and ceramide generation, as well as with activation of stress-related kinases, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK) pathways. We also provide evidence for the participation of JNK and p38 in ethanol-induced cell death, because pharmacological inhibitors of these kinases largely prevent the apoptosis induced by ethanol or by ethanol and C2-ceramide. Furthermore, we show that ethanol-induced ERK activation triggers the stimulation of cyclo-oxygenase-2 (COX-2) and the release of prostaglandin E2, and that blockade of the mitogen-activated protein kinase kinase (MEK)/ERK pathway by PD98059 abolishes the up-regulation of COX-2 induced by ethanol plus ceramide, and decreases the ethanol-induced apoptosis. These results strongly suggest that ethanol is able to stimulate the SMase–ceramide pathway, leading to the activation of signalling pathways implicated in cell death. These findings provide an insight into the mechanisms involved in ethanol-induced astroglial cell death during brain development.
The role of apoptosis in EtOH-induced liver injury has not been investigated much. Therefore, the question whether apoptosis is a contributory factor to alcoholic liver disease remains to be answered. The purpose of this study was to characterize the liver apoptotic response in a murine model of alcohol-enhanced lipopolysaccharide (LPS) hepatotoxicity.
Mice were fed an alcohol-containing liquid diet for 49 days followed by an acute LPS challenge. The liver state was judged on the basis of histological appearance, plasma liver enzyme activity (alanine:2-oxoglutarate and aspartate:2-oxoglutarate aminotransferases, as markers of hepatocytolysis), and plasma hyaluronan levels (as a marker of the sinusoidal endothelial cell scavenging function). The liver apoptotic response was assessed by DNA fragmentation (TUNEL procedure), and caspases-3 and -8 activity. To determine if ceramide played a role in the liver apoptotic response, the activity of acidic sphingomyelinase and tissue content of ceramide were also quantified.
Alcohol exposure induced fat accumulation and sensitized the liver to LPS injurious effects. Plasma liver enzyme activity was elevated by alcohol and this effect was potentiated by LPS. Liver apoptosis was augmented by both alcohol and LPS treatment as reflected by high frequency of positive TUNEL staining nuclei and by an increased activity of caspase-3 and -8. Acidic sphingomyelinase activity was also increased and it was associated with an elevated tissue content of ceramide. In addition, LPS also increased plasma TNF-α levels. These changes were accompanied by elevated plasma hyaluronan, reflecting an impaired sinusoidal endothelial cell scavenging function.
These results provide a more complete description of the liver apoptotic response to both alcohol and LPS and may constitute the basis for further mechanistic studies on a possible role apoptosis may play in alcoholic liver injury.
Acid sphingomyelinase occupies a prominent position in sphingolipid catabolism, catalyzing the hydrolysis of sphingomyelin to ceramide and phosphorylcholine. Enzymatic dysfunction of acid sphingomyelinase results in Niemann–Pick disease, a lysosomal storage disorder characterized at the cellular level by accumulation of sphingomyelin within the endo-lysosomal compartment. Over the past decade interest in the role of acid sphingomyelinase has moved beyond its ‘housekeeping’ function in constitutive turnover of sphingomyelin in the lysosome to include study of regulated ceramide generation. Ceramide functions as a bioactive sphingolipid with pleiotropic signaling properties, and has been implicated in diverse cellular processes of physiologic and pathophysiologic importance. Though many cellular enzymes have the capacity to generate ceramide, there is growing appreciation that ‘all ceramides are not created equal.’ Ceramides likely exert distinct effects in different cellular/subcellular compartments by virtue of access to other sphingolipid enzymes (e.g. ceramidases), effector molecules (e.g. ceramide-activated protein phosphatases), and neighboring lipids and proteins (e.g. cholesterol, ion channels). One of the unique features of acid sphingomyelinase is that it has been implicated in the hydrolysis of sphingomyelin in three different settings — the endo-lysosomal compartment, the outer leaflet of the plasma membrane, and lipoproteins. How a single gene product has the capacity to function in these diverse settings, and the subsequent impact on downstream ceramide-mediated biology is the subject of this review.
An improved method for the detection of Niemann-Pick disease type A and B carriers is described. Niemann-Pick disease is a genetic disorder characterized by the accumulation of sphingomyelin in several organs, among them spleen, liver and brain. A deficiency in sphingomyelinase activity is the characteristic defect in this lipid storage disorder, and the decreased enzyme activity in these types can be demonstrated in various cells and tissues. Low activity levels of enzymes were found in leukocyte fractions of peripheral blood from patients screened for Niemann-Pick disease. In heterozygote patients levels were within the lower range of control values. We therefore determined sphingomyelinase activity in discrete cell types isolated by two different methods from the total leukocyte fraction. In normal subjects sphingomyelinase activity is five to ten-fold higher in lymphocytes than in granulocytes. Monocytes have an intermediate level. pH profile and stability to heat inactivation were tested in separated lymphocytes and granulocytes and were found to be insignificantly different. Detection of carriers for Niemann-Pick type A and B by using separated lymphocyte as enzyme source, overcomes the difficulty of overlapping values with normal controls and serves as an excellent tool to detect heterozygotes.
Ceramide, produced through either the induction of SM hydrolysis or synthesized de novo transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions. A generalized ceramide signal transduction scheme is shown in Fig. 2 in which ceramide is generated through the activation of distinct SMases residing in separate subcellular compartments in response to specific stimuli. Clearly, specificity of cellular responses to ceramide depends upon many factors which include the nature of the stimulus, co-stimulatory signals and the cell type involved. Ceramide derived from neutral SMase activation is thought to be involved in modulating CAPK and MAP kinases, PLA2 (arachidonic acid mobilization), and CAPP while ceramide generated through acid SMase activation appears to be primarily involved in NF-kappa B activation. While there is no apparent cross-talk between these two ceramide-mediated signalling pathways, there is likely to be significant cross-talk between ceramide signalling and other signal transduction pathways (e.g., the PKC and MAP kinase pathways). Other downstream targets for ceramide action include Cox, IL-6 and IL-2 gene expression, PKC zeta, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators. Many, if not all, of these ceramide-mediated signalling events have been identified in the various cells comprising the immune system and are integral to the optimal functioning of the immune system. Although the role of the SM pathway and the generation of ceramide in T and B lymphocytes have only recently been recognized, it is clear from these studies that signal transduction through SM and ceramide can strongly affect the immune response, either directly through cell signalling events, or indirectly through cytokines produced by other cells as the result of signalling through the SM pathway. An overview of the signalling mechanisms coupling ceramide to the modulation of the immune response is depicted in Fig. 3 and shows how ceramide may play pivotal roles in regulating a number of complex processes. The SM pathway represents a potentially valuable focal point for therapeutic control of immune responses, perhaps for either enhancement of the activity of T cells in the elimination of tumors, or the down-regulation of lymphocyte function in instances of autoimmune disease. The recent explosion of knowledge regarding ceramide signalling notwithstanding, a number of critical questions need to be answered before a comprehensive, mechanistic understanding can be formulated relative to the incredibly varied effects of ceramide on cell function. For example, (i) how is a structurally simple molecule like ceramide able to mediate so many different, and sometimes paradoxical, physiological responses ranging from cell proliferation and differentiation to inhibition of cell growth and apoptosis, (ii) what are the molecular identities and modes of activation of the various SMase isoforms, (iii) what determines the distribution of the unique isoforms of SMase in cells of different lineages or at different stages of differentiation, (iv) what is the relative contribution of ceramide generated through SM hydrolysis versus de novo synthesis, and (v) by what means does ceramide interact with specific intracellular targets? Although a number of ceramide-activatable kinases, phosphatases, and their protein substrates have been identified, a more extensive search for additional cellular targets will be indispensable in determining the phosphorylation cascades linking the activation of the SM pathway to the regulation of nuclear events. Clearly, cross-talk between ceramide-induced signal transduction cascades and other signalling pathways adds to the inherent difficulty in distinguishing the specific effects of complex, intertwining signalling pathways.
Stress is believed to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response. Conclusive evidence for this paradigm, however, is lacking. In the present study, we used a genetic approach to address this issue directly. We show that lymphoblasts from Niemann-Pick patients, which have an inherited deficiency of acid sphingomyelinase activity, fail to respond to ionizing radiation with ceramide generation and apoptosis. These abnormalities are reversible up on restoration of acid sphingomyelinase activity by retroviral transfer of human acid sphingomyelinase cDNA. Acid sphingomyelinase knockout mice also expressed defects in radiation-induced ceramide generation and apoptosis in vivo. Comparison with p53 knockout mice revealed that acid sphingomyelinase-mediated apoptosis and p53-mediated apoptosis are likely distinct and independent. These genetic models provide definitive evidence for the involvement of acid sphingomyelinase in one form of stress-induced apoptosis.
The role of apoptosis in EtOH-induced liver injury has not been investigated much. Therefore, the question whether apoptosis is a contributory factor to alcoholic liver disease remains to be answered. The purpose of this study was to characterize the liver apoptotic response in a murine model of alcohol-enhanced lipopolysaccharide (LPS) hepatotoxicity.
Mice were fed an alcohol-containing liquid diet for 49 days followed by an acute LPS challenge. The liver state was judged on the basis of histological appearance, plasma liver enzyme activity (alanine:2-oxoglutarate and aspartate:2-oxoglutarate aminotransferases, as markers of hepatocytolysis), and plasma hyaluronan levels (as a marker of the sinusoidal endothelial cell scavenging function). The liver apoptotic response was assessed by DNA fragmentation (TUNEL procedure), and caspases-3 and -8 activity. To determine if ceramide played a role in the liver apoptotic response, the activity of acidic sphingomyelinase and tissue content of ceramide were also quantified.
Alcohol exposure induced fat accumulation and sensitized the liver to LPS injurious effects. Plasma liver enzyme activity was elevated by alcohol and this effect was potentiated by LPS. Liver apoptosis was augmented by both alcohol and LPS treatment as reflected by high frequency of positive TUNEL staining nuclei and by an increased activity of caspase-3 and -8. Acidic sphingomyelinase activity was also increased and it was associated with an elevated tissue content of ceramide. In addition, LPS also increased plasma TNF-alpha levels. These changes were accompanied by elevated plasma hyaluronan, reflecting an impaired sinusoidal endothelial cell scavenging function.
These results provide a more complete description of the liver apoptotic response to both alcohol and LPS and may constitute the basis for further mechanistic studies on a possible role apoptosis may play in alcoholic liver injury.
Many receptor systems use receptor clustering for transmembrane signaling. In this study, we show that acid sphingomyelinase (ASM) is essential for the clustering of CD40. Stimulation of lymphocytes via CD40 ligation results in ASM translocation from intracellular stores, most likely vesicles, into distinct membrane domains on the extracellular surface of the plasma membrane. Surface ASM initiates a release of extracellularly oriented ceramide, which in turn mediates CD40 clustering in sphingolipid-rich membrane domains. ASM, ceramide, and CD40 colocalize in the cap-like structure of stimulated cells. Deficiency of ASM, destruction of sphingolipid-rich rafts, or neutralization of surface ceramide prevents CD40 clustering and CD40-initiated cell signaling. These findings indicate that the ASM-mediated release of ceramide and/or metabolites of ceramide regulate clustering of CD40, which seems to be a prerequisite for cellular activation via CD40.
Impaired health caused by alcohol abuse has been known throughout recorded history. Over the past century, alcohol abuse has been clearly linked to host susceptibility to infectious disease, particularly bacterial pneumonia. Recently, both acute and chronic alcohol intake have been shown to result in specific defects in innate and adaptive immunity; these could, in principle, be subjected to specific modulation to overcome the immunosuppressive effects of the most commonly abused substance in the Western world.
This paper reviews our present knowledge of sphingomyelinases as enzymes, and as enzymes acting on a membrane constituent lipid, sphingomyelin. Six types of sphingomyelinases are considered, namely acidic, secretory, Mg(2+)-dependent neutral, Mg(2+)-independent neutral, alkaline, and bacterial enzymes with both phospholipase C and sphingomyelinase activity. Sphingomyelinase assay methods and specific inhibitors are reviewed. Kinetic and mechanistic studies are summarized, a kinetic model and a general-base catalytic mechanism are proposed. Sphingomyelinase-membrane interactions are considered from the point of view of the influence of lipids on the enzyme activity. Moreover, effects of sphingomyelinase activity on membrane architecture (increased membrane permeability, membrane aggregation and fusion) are described. Finally, a number of open questions on the above topics are enunciated.
Early events required for induction of apoptosis by CD95 are preassociation of CD95, the formation of the death-inducing signaling complex (DISC) and clustering of CD95 in distinct membrane domains. Here, we identify the molecular ordering of these events and show that the acid sphingomyelinase (ASM) functions upstream of the DISC to mediate CD95 clustering in ceramide-enriched membrane platforms, an event that is required for DISC formation. Experiments in ASM-deficient cells revealed that CD95 ligation, in the absence of ceramide generation, triggers <1% of full caspase 8 activation at the receptor. This event, however, is both necessary and sufficient to trigger translocation of ASM onto the outer leaflet of the plasma membrane, ASM activation and ceramide release, but insufficient for apoptosis induction. Ceramide-mediated CD95 clustering then amplifies the primary CD95 signaling and drives the second step of CD95 signaling, that is, formation of the DISC yielding 100% caspase activity and apoptosis. These studies suggest that the most parsimonious interpretation of the molecular ordering of the earliest events in CD95 signaling, at least in some cells, is: CD95 ligation-->1% of maximum caspase 8 activation-->ASM translocation-->ceramide generation-->CD95 clustering-->DISC formation-->100% of maximum caspase 8 activation-->apoptosis.
Major depressive disorder (MDD) is highly co-morbid with other Axis I disorders, which commonly precede its onset. We sought to determine the level and periods of risk for MDD posed by prior or co-occurring psychiatric disorders.
Using retrospective data from a longitudinal, population-based sample of 2926 male and 1929 female adult twin subjects, we predicted the hazard rates for MDD from a Cox proportional hazards model with same-year or prior onsets of co-morbid Axis I disorders as time-dependent covariates.
All axis I disorders studied (generalized anxiety disorder, panic disorder, phobia, alcohol dependence, psychoactive substance use disorders and conduct disorder) significantly predicted increased risk for developing MDD. The highest hazard rates occurred for MDD onsets that co-occurred with those of the co-morbid disorder. However, the risk for onset of MDD subsequent to that of prior disorders is also significantly increased and remains relatively unchanged over time. Although the risk for onset of MDD is significantly higher in women than men, this was not explained by gender differences in prior disorder prevalence or increased sensitivity in women to the effects of prior disorders on risk for depression.
Prior psychiatric disorders are significant risk factors for the development of MDD, independent of the length of the intervening period between the onset of the first disorder and that of MDD.
Ethanol induces apoptosis in cultured neurons. To assess the involvement of sphingolipids and neutral lipids in the apoptotic process, ethanol-induced alterations in lipid content and metabolism were examined by using primary cultured rat cerebellar granule neurons (CGNs), human neuroblastoma SK-N-SH cells, and mouse neuroblastoma Neuro2a cells. Ethanol treatment conditions that induced apoptosis in CGNs and SK-N-SH cells but not in Neuro2a cells were used for these experiments.
Cultured neurons were treated with and without 100 mM ethanol for one to three days, and the amounts of cellular sphingolipids [ceramide, glucosylceramide (GlcCer), and sphingomyelin] and neutral lipids [cholesterol, triglyceride (TG), and cholesterol ester (ChE)] were analyzed by high-performance thin-layer chromatography, using a Coomassie brilliant blue staining method. The incorporation of [C] acetate into each lipid fraction was measured in CGNs treated with and without ethanol. Also, the effect of delipidated serum, sterols, myriocin (a serine-palmitoyltransferase inhibitor), and desipramine (an acid sphingomyelinase inhibitor) on ethanol-induced lipid changes was studied by using Neuro2a cells.
The most prominent change common to CGN, SK-N-SH, and Neuro2a cells was ethanol-induced TG accumulation. Higher incorporation of radioactivity into TG was also observed in ethanol-treated cultures when cellular lipids were metabolically labeled with [C] acetate in CGNs. In addition, ethanol elevated ceramide levels in all these neurons. However, ethanol induced decreases in GlcCer along with the reduction of cell viability in SK-N-SH cells and CGNs, whereas it increased GlcCer in Neuro2a cells that remained viable. Myriocin, which reduced ceramide levels, attenuated ethanol-induced cell death in SK-N-SH cells. Ethanol-induced accumulation of TG was sterol-independent, whereas changes in ceramide and GlcCer were affected in Neuro2a cells by the presence of sterols in the medium. Staurosporine, which induced cell death in SK-N-SH cells, increased levels of TG, ChE, and ceramides and reduced the level of GlcCer.
The results showing that ethanol induces accumulation of TG and ceramide in cultured neurons suggest that ethanol enhances lipogenesis and/or reduces fatty acid degradation in neurons, as previously observed in other cell types. Further, ethanol-induced changes in lipid metabolism, specifically those of ceramide and GlcCer, may be related to the ethanol-induced apoptotic pathway.
Sphingolipids including ceramide and its derivatives such as ceramide-1-phosphate, glycosyl-ceramide, and sphinogosine (-1-phosphate) are now recognized as novel intracellular signal mediators for regulation of inflammation, apoptosis, proliferation, and differentiation. One of the important and regulated steps in these events is the generation of these sphingolipids via hydrolysis of sphingomyelin through the action of sphingomyelinases (SMase). Several lines of evidence suggest that reactive oxygen species (ROS; O2-, H2O2, and OH-,) and reactive nitrogen species (RNS; NO, and ONOO-) and cellular redox potential, which is mainly regulated by cellular glutathione (GSH), are tightly linked to the regulation of SMase activation. On the other hand, sphingolipids are also known to play an important role in maintaining cellular redox homeostasis through regulation of NADPH oxidase, mitochondrial integrity, and antioxidant enzymes. Therefore, this paper reviews the relationship between cellular redox and sphingolipid metabolism and its biological significance.
Unlabelled:
Ethanol abuse is one of the major etiologies of cirrhosis. Ethanol has been shown to induce apoptosis via activation of oxidative stress, mitogen-activated protein kinases (MAPK), and tyrosine kinases. However, there is a paucity of data that examine the interplay among these molecules. In the present study we have systematically elucidated the role of novel protein kinase C isoforms (nPKC; PKCdelta and PKCepsilon) in ethanol-induced apoptosis in hepatocytes. Ethanol enhanced membrane translocation of PKCdelta and PKCepsilon, which was associated with the phosphorylation of p38MAPK, p42/44MAPK and JNK1/2, and the nuclear translocation of NF-kappaB and AP-1. This resulted in increased apoptosis in primary rat hepatocytes. Inhibition of both PKCdelta and PKCepsilon resulted in a decreased MAPK activation, decreased nuclear translocation of NF-kappaB and AP-1, and inhibition of apoptosis. In addition, ethanol activated the tyrosine phosphorylation of PKCdelta via tyrosine kinase in hepatocytes. The tyrosine phosphorylated PKCdelta was cleaved by caspase-3 and these fragments were translocated to the nucleus. Inhibition of ethanol-induced oxidative stress blocked the membrane translocation of PKCdelta and PKCepsilon, and the tyrosine phosphorylation of PKCdelta in hepatocytes. Inhibition of oxidative stress, tyrosine kinase or caspase-3 activity caused a decreased nuclear translocation of PKCdelta in response to ethanol, and was associated with less apoptosis.
Conclusion:
These results provide a newly-described mechanism by which ethanol induces apoptosis via activation of nPKC isoforms in hepatocytes.
Some organic weak bases induce a detachment from inner lysosomal membranes and subsequent inactivation of acid sphingomyelinase (ASM) and thus work as functional ASM inhibitors. The aim of the present investigation was to develop a structure-property-activity relation (SPAR) model in order to specify the structural and physicochemical characteristics of probes capable of functionally inhibiting ASM. High p K a and high log P values are necessary but not sufficient preconditions for functional inhibition of ASM. The experimental data supported the requirement of an additional factor, which is necessary for functional inhibition of ASM. This factor k is related to the steric hindrance of the most basic nitrogen atom and presumably modulates the free presentation of a protonated nitrogen atom at the inner lysosomal surface. During the course of the study, we characterized 26 new functional ASM inhibitors, including doxepine 63, fluoxetine 104, maprotiline 109, nortriptyline 114, paroxetine 118, sertraline 124, suloctidil 125, and terfenadine 127.
The pathogenesis of alcohol-induced liver disease (ALD) is still poorly understood. One of the clues to its progression relates to the alcohol-mediated susceptibility of hepatocytes to cell death by reactive oxygen species (ROS) and inflammatory cytokines. Tumor necrosis factor alpha (TNF) has been considered a key ALD mediator with acidic sphingomyelinase (ASMase)-mediated ceramide generation playing a critical role. TNF receptor 1 and 2 knock-out mice or ASMase(-/-) mice exhibit resistance to alcohol-mediated fatty liver and cell death. Furthermore, alcohol feeding has been shown to sensitize hepatocytes to TNF due to the limitation of mitochondrial glutathione (mGSH) through impaired import of GSH from the cytosol due to altered membrane order parameter caused by mitochondrial cholesterol increase. Selective pharmacological depletion of mGSH sensitizes hepatocytes to TNF-mediated cell death, which reproduces the observations found with alcohol feeding. TNF signaling analyses in hepatocytes with or without mGSH depletion indicate that mGSH prevents cardiolipin peroxidation (CLOOH) formation by TNF-induced ROS via ASMase and that CLOOH cooperates with oligomerized Bax to cause mitochondrial outer membrane permeabilization through destabilization of the lipid bilayer via increased bilayer-to-inverted hexagonal phase transitions. Thus, activation of ASMase and cholesterol-mediated mGSH depletion both collaborate to promote alcohol-induced TNF-mediated hepatocellular damage, suggesting novel therapeutic opportunities in ALD.
Acid sphingomyelinase (ASM; E.C. 3.1.4.12) is best known for its involvement in the lysosomal storage disorder Niemann-Pick disease (NPD). Through studies that began by investigating this rare disease, recent findings have uncovered the important role of this enzyme in the initiation of ceramide-mediated signal transduction. This unique function involves translocation of the enzyme from intracellular compartments to the outer leaflet of the cell membrane, where hydrolysis of sphingomyelin into ceramide initiates membrane reorganization and facilitates the formation and coalescence of lipid microdomains. These microdomains are sites of protein-protein interactions that lead to downstream signaling, and perturbation of microdomain formation influences the pathophysiology of many common diseases. The initial observations implicating ASM in this process have come from studies using cells from patients with NPD or from ASM knockout (ASMKO) mice, where the genetic deficiency of this enzymatic activity has been shown to protect these cells and animals from stress-induced and developmental apoptosis. This review will discuss the complex biology of this enzyme in the context of these new findings and its recently reported importance in common human diseases, including cancer, sepsis, cardiovascular, pulmonary, liver, and neurological diseases as well as the potential for using ASM (or ASM inhibitors) as therapeutic agents.