Sphingosine induction of the Pseudomonas aeruginosa hemolytic phospholipase C/sphingomyelinase (PlcH).

Hemolytic phospholipase C, PlcH, is an important virulence factor for Pseudomonas aeruginosa. PlcH preferentially hydrolyzes sphingomyelin and phosphatidylcholine, and this hydrolysis activity drives tissue damage and inflammation and interferes with the oxidative burst of immune cells. Among other contributors, transcription of plcH was previously shown to be induced by phosphate starvation via PhoB and the choline metabolite, glycine betaine, via GbdR. Here, we show that sphingosine can induce plcH transcription and result in secreted PlcH enzyme activity. This induction is dependent on the sphingosine-sensing transcriptional regulator SphR. The SphR induction of plcH occurs from the promoter for the gene upstream of plcH that encodes the neutral ceramidase, CerN, and transcriptional readthrough of the cerN transcription terminator. Evidence for these conclusions came from mutation of the SphR binding site in the cerN promoter, mutation of the cerN terminator, enhancement of cerN termination by adding the rrnB terminator, and reverse transcriptase PCR (RT-PCR) showing that the intergenic region between cerN and plcH is made as RNA during sphingosine, but not choline, induction. We also observed that, like glycine betaine induction, sphingosine induction of plcH is under catabolite repression control, which likely explains why such induction was not seen in other studies using sphingosine in rich media. The addition of sphingosine as a novel inducer for PlcH points to the regulation of plcH transcription as a site for the integration of multiple host-derived signals. IMPORTANCE: PlcH is a secreted phospholipase C/sphingomyelinase that is important for the virulence of Pseudomonas aeruginosa. Here, we show that sphingosine, which presents itself or as a product of P. aeruginosa sphingomyelinase and ceramidase activity, leads to the induction of plcH transcription. This transcriptional induction occurs from the promoter of the upstream ceramidase gene generating a conditional operon. The transcript on which plcH resides, therefore, is different depending on which host molecule or condition leads to induction, and this may have implications for PlcH post-transcriptional regulation. This work also adds to our understanding of P. aeruginosa with host-derived sphingolipids.

Efficacy of ceramidase inhibition on human renal cell carcinoma: a cell culture study.

OBJECTIVE: Cancer-preventative medicines like curcumin, resveratrol, and nonsteroidal anti-inflammatory medications all have their effects modulated by ceramide. According to research, these medications raise ceramide levels in cancer cells, leading to programmed cell death. Recently, cancer research has been involved in sphingolipid metabolism. The critical molecule here is ceramide. We aimed to investigate if the inhibition of ceramidases induces death in the human renal cell carcinoma cell line. MATERIALS AND METHODS: Human kidney carcinoma A-498 (ATCC® HTB-44™) cells were used as test cells. Ceranib-2, fetal bovine serum (FBS), penicillin/streptomycin, dimethyl sulfoxide, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium bromide and Dulbecco's Modified Eagle Medium High Glucose, caspase 3/7, annexin-V, Bcl-2 activation dual detection, and MitoPotential kits were used. 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay, annexin-V analysis, caspase 3/7 analysis, Bcl-2 activation analysis, and measurement of mitochondrial membrane potential were performed. RESULTS: MTT colorimetric assay results for 24 hours indicated that the viability of human renal cell carcinoma cells decreased compared to the control group with an increase in the applied concentration of the ceramidase inhibitor-ceranib-2. The growth inhibition by ceranib-2 for 24 hours did not decrease the viability under 50%; thus, it could not be possible to calculate the IC50 value for the short-term application of ceranib-2 for 24 hours to A-498 cells. A statistically significant decrease in cell viability was recorded at doses of 100, 50, 25, and 12.2 µM of ceranib-2, and no significant decrease was detected at the lower doses of ceranib-2. The highest inhibition caused by ceranib-2 on human renal cell carcinoma cells A-498 was detected at an application time of 72 hours. This inhibition was statistically significant for all applied doses of ceranib-2 on A-498 cells compared to untreated cells. Annexin-V technique that detects the translocation of phosphatidylserine to the outer membrane of apoptotic cells indicated that after the application of ceranib-2, apoptosis was triggered on A-498 cells with a total apoptotic profile of 12.12% compared to the untreated cells that were used as controls. Compared to untreated A-498 cells, a rise in percentage to 16.25% of cells with activated caspases 3/7 was recorded after applying IC50 concentration of ceranib-2 on A-498 cells for 48 hours. CONCLUSIONS: The results of our study indicated that the application of ceramidase inhibitor, ceranib-2 on human renal cell carcinoma A-498 cells cause cytotoxicity, antiproliferative, growth inhibitory, and apoptotic efficacies in a dose and time-dependent manner probably via inhibiting the acid ceramidases that hydrolyze ceramides that induce cell death. For further conclusions, more mechanical, pharmacokinetic, and pharmaceutic, as well as in vitro and in vivo anti-cancer activity investigations are required.

Development of Comorbid Depression after Social Fear Conditioning in Mice and Its Effects on Brain Sphingolipid Metabolism.

Currently, there are no animal models for studying both specific social fear and social fear with comorbidities. Here, we investigated whether social fear conditioning (SFC), an animal model with face, predictive and construct validity for social anxiety disorder (SAD), leads to the development of comorbidities at a later stage over the course of the disease and how this affects the brain sphingolipid metabolism. SFC altered both the emotional behavior and the brain sphingolipid metabolism in a time-point-dependent manner. While social fear was not accompanied by changes in non-social anxiety-like and depressive-like behavior for at least two to three weeks, a comorbid depressive-like behavior developed five weeks after SFC. These different pathologies were accompanied by different alterations in the brain sphingolipid metabolism. Specific social fear was accompanied by increased activity of ceramidases in the ventral hippocampus and ventral mesencephalon and by small changes in sphingolipid levels in the dorsal hippocampus. Social fear with comorbid depression, however, altered the activity of sphingomyelinases and ceramidases as well as the sphingolipid levels and sphingolipid ratios in most of the investigated brain regions. This suggests that changes in the brain sphingolipid metabolism might be related to the short- and long-term pathophysiology of SAD.

Sex Differences in Sphingosine-1-Phosphate Levels Are Dependent on Ceramide Synthase 1 and Ceramidase in Lung Physiology and Tumor Conditions.

Sex is a biological variable that can reflect clinical outcomes in terms of quality of life, therapy effectiveness, responsiveness and/or toxicity. Sphingosine-1-phosphate (S1P) is a lipidic mediator whose activity can be influenced by sex. To evaluate whether the S1P axis underlies sex 'instructions' in the lung during physiological and oncological lung conditions, sphingosine and S1P were quantified in the blood of healthy (H) volunteers, lung adenocarcinoma (ADK) and squamous cell carcinoma (SCC) patients of both sexes. S1P receptors and their metabolic enzymes were evaluated in the tissues. Circulating levels of S1P were similar among H female and male subjects and female SCC patients. Instead, male and female ADK patients had lower circulating S1P levels. S1P receptor 3 (S1PR3) was physiologically expressed in the lung, but it was overexpressed in male SCC, and female and male ADK, but not in female SCC patients, who showed a significantly reduced ceramide synthase 1 (CERS1) mRNA and an overexpression of the ceramidase (ASAH1) precursor in lung tumor tissues, compared to male SCC and both male and female ADK patients. These findings highlighted sex differences in S1P rheostat in pathological conditions, but not in physiological conditions, identifying S1P as a prognostic mediator depending on lung cancer histotype.

The Dark Side of Sphingolipids: Searching for Potential Cardiovascular Biomarkers.

Cardiovascular diseases (CVDs) are the leading cause of death and illness in Europe and worldwide, responsible for a staggering 47% of deaths in Europe. Over the past few years, there has been increasing evidence pointing to bioactive sphingolipids as drivers of CVDs. Among them, most studies place emphasis on the cardiovascular effect of ceramides and sphingosine-1-phosphate (S1P), reporting correlation between their aberrant expression and CVD risk factors. In experimental in vivo models, pharmacological inhibition of de novo ceramide synthesis averts the development of diabetes, atherosclerosis, hypertension and heart failure. In humans, levels of circulating sphingolipids have been suggested as prognostic indicators for a broad spectrum of diseases. This article provides a comprehensive review of sphingolipids' contribution to cardiovascular, cerebrovascular and metabolic diseases, focusing on the latest experimental and clinical findings. Cumulatively, these studies indicate that monitoring sphingolipid level alterations could allow for better assessment of cardiovascular disease progression and/or severity, and also suggest them as a potential target for future therapeutic intervention. Some approaches may include the down-regulation of specific sphingolipid species levels in the circulation, by inhibiting critical enzymes that catalyze ceramide metabolism, such as ceramidases, sphingomyelinases and sphingosine kinases. Therefore, manipulation of the sphingolipid pathway may be a promising strategy for the treatment of cardio- and cerebrovascular diseases.

Targeting Src reactivates pyroptosis to reverse chemoresistance in lung and pancreatic cancer models.

Pancreatic and lung cancers frequently develop resistance to chemotherapy-induced cell apoptosis during the treatment, indicating that targeting nonapoptotic-related pathways, such as pyroptosis, can be an alternative cancer treatment strategy. Pyroptosis is a gasdermin-driven lytic programmed cell death triggered by inflammatory caspases when initiated by canonical or noncanonical pathways that has been recently seen as a potential therapeutic target in cancer treatment. However, overcoming chemoresistance in cancers by modulating pyroptosis has not been explored. Here, we demonstrate that ß5-integrin represses chemotherapy-induced canonical pyroptosis to confer cancer chemoresistance through ASAH2-driven sphingolipid metabolic reprogramming. Clinically, high ß5-integrin expression associates with poor patient prognosis and chemotherapeutic responses in cancers. In addition, chemoresistant cells in vitro fail to undergo chemotherapy-induced pyroptosis, which is controlled by ß5-integrin. Mechanistically, proteomic and lipidomic analyses indicate that ß5-integrin up-regulates sphingolipid metabolic enzyme ceramidase (ASAH2) expression through Src-signal transducer and activator of transcription 3 (STAT3) signaling, which then reduces the metabolite ceramide concentration and subsequent ROS production to prohibit chemotherapy-induced canonical pyroptosis. Using cancer cell lines, patient-derived tumor organoids, and orthotopic lung and pancreatic animal models, we show that administration of a Src or ceramidase inhibitor rescues the response of chemoresistant pancreatic and lung cancer cells to chemotherapy by reactivating pyroptosis in vitro and in vivo. Overall, our results suggest that pyroptosis-based therapy is a means to improve cancer treatment and warrants further investigation.

Alkaline ceramidase catalyzes the hydrolysis of ceramides via a catalytic mechanism shared by Zn2+-dependent amidases.

Human alkaline ceramidase 3 (ACER3) is one of three alkaline ceramidases (ACERs) that catalyze the conversion of ceramide to sphingosine. ACERs are members of the CREST superfamily of integral-membrane hydrolases. All CREST members conserve a set of three Histidine, one Aspartate, and one Serine residue. Although the structure of ACER3 was recently reported, catalytic roles for these residues have not been biochemically tested. Here, we use ACER3 as a prototype enzyme to gain insight into this unique class of enzymes. Recombinant ACER3 was expressed in yeast mutant cells that lack endogenous ceramidase activity, and microsomes were used for biochemical characterization. Six-point mutants of the conserved CREST motif were developed that form a Zn-binding active site based on a recent crystal structure of human ACER3. Five point mutants completely lost their activity, with the exception of S77A, which showed a 600-fold decrease compared with the wild-type enzyme. The activity of S77C mutant was pH sensitive, with neutral pH partially recovering ACER3 activity. This suggested a role for S77 in stabilizing the oxyanion of the transition state. Together, these data indicate that ACER3 is a Zn2+-dependent amidase that catalyzes hydrolysis of ceramides via a similar mechanism to other soluble Zn-based amidases. Consistent with this notion, ACER3 was specifically inhibited by trichostatin A, a strong zinc chelator.

Mechanistic insights into ceramidase inhibitor LCL521-enhanced tumor cell killing by photodynamic and thermal ablation therapies.

Our recent investigation uncovered that the acid ceramidase inhibitor LCL521 enhances the direct tumor cell killing effect of photodynamic therapy (PDT) treatment. The present study aimed at elucidating the mechanisms underlying this effect. Exposing mouse squamous cell carcinoma SCCVII cells treated with temoporfin-based PDT to LCL521 (rising ceramide concentration) produced a much greater decrease in cell survival than comparable exposure to the sphingosine kinase-1 inhibitor PF543 (that reduces sphingosine-1-phosphate concentration). This is consistent with recognizing the rising levels of pro-apoptotic sphingolipid ceramide as being more critical in promoting the death of PDT-treated cells than the reduction in the availability of pro-survival acting sphingosine-1 phosphate. This pro-apoptotic impact of LCL521, which was suppressed by the apoptosis inhibitor bongkrekic acid, involves the interaction with the cellular stress signaling network. Hence, inhibiting the key elements of these pathways markedly influenced the adjuvant effect of LCL521 on the PDT response. Particularly effective was the inositol-requiring element-1 (IRE1) kinase inhibitor STF-083010 that dramatically enhanced the killing of cells treated with PDT plus LCL521. An important role in the survival of these cells was exhibited by master transcription factors STAT3 and HIF-1α. The STAT3 inhibitor NSC 74859 was especially effective in further reducing the cell survival rates, suggesting its possible exploitation for therapeutic gain. An additional finding in this study is that LCL521-promoted PDT-mediated cell killing through ceramide-mediated lethal effects is extended to the interaction with other cancer treatment modalities with a rapid cellular stress impact such as photothermal therapy (PTT) and cryoablation therapy (CAT).

Elusive Roles of the Different Ceramidases in Human Health, Pathophysiology, and Tissue Regeneration.

Ceramide and sphingosine are important interconvertible sphingolipid metabolites which govern various signaling pathways related to different aspects of cell survival and senescence. The conversion of ceramide into sphingosine is mediated by ceramidases. Altogether, five human ceramidases-named acid ceramidase, neutral ceramidase, alkaline ceramidase 1, alkaline ceramidase 2, and alkaline ceramidase 3-have been identified as having maximal activities in acidic, neutral, and alkaline environments, respectively. All five ceramidases have received increased attention for their implications in various diseases, including cancer, Alzheimer's disease, and Farber disease. Furthermore, the potential anti-inflammatory and anti-apoptotic effects of ceramidases in host cells exposed to pathogenic bacteria and viruses have also been demonstrated. While ceramidases have been a subject of study in recent decades, our knowledge of their pathophysiology remains limited. Thus, this review provides a critical evaluation and interpretive analysis of existing literature on the role of acid, neutral, and alkaline ceramidases in relation to human health and various diseases, including cancer, neurodegenerative diseases, and infectious diseases. In addition, the essential impact of ceramidases on tissue regeneration, as well as their usefulness in enzyme replacement therapy, is also discussed.

Fingolimod Affects Transcription of Genes Encoding Enzymes of Ceramide Metabolism in Animal Model of Alzheimer's Disease.

The imbalance in sphingolipid signaling may be critically linked to the upstream events in the neurodegenerative cascade of Alzheimer's disease (AD). We analyzed the influence of mutant (V717I) amyloid ß precursor protein (AßPP) transgene on sphingolipid metabolism enzymes in mouse hippocampus. At 3 months of age AßPP/Aß presence upregulated enzymes of ceramide turnover on the salvage pathway: ceramide synthases (CERS2, CERS4, CERS6) and also ceramidase ACER3. At 6 months, only CERS6 was elevated, and no ceramide synthase was increased at 12 months. However, sphingomyelin synthases, which utilize ceramide on the sphingomyelinase pathway, were reduced (SGMS1 at 12 and SGMS2 at 6 months). mRNAs for sphingomyelin synthases SGMS1 and SGMS2 were also significantly downregulated in human AD hippocampus and neocortex when compared with age-matched controls. Our findings suggest early-phase deregulation of sphingolipid homeostasis in favor of ceramide signaling. Fingolimod (FTY720), a modulator of sphingosine-1-phosphate receptors countered the AßPP-dependent upregulation of hippocampal ceramide synthase CERS2 at 3 months. Moreover, at 12 months, FTY720 increased enzymes of ceramide-sphingosine turnover: CERS4, ASAH1, and ACER3. We also observed influence of fingolimod on the expression of the sphingomyelinase pathway enzymes. FTY720 counteracted the AßPP-linked reduction of sphingomyelin synthases SGMS1/2 (at 12 and 6 months, respectively) and led to elevation of sphingomyelinase SMPD2 (at 6 and 12 months). Therefore, our results demonstrate potentially beneficial, age-specific effects of fingolimod on transcription of sphingolipid metabolism enzymes in an animal model of AD.

Golgi-Localized PAQR4 Mediates Antiapoptotic Ceramidase Activity in Breast Cancer.

The metabolic network of sphingolipids plays important roles in cancer biology. Prominent sphingolipids include ceramides and sphingosine-1-phosphate that regulate multiple aspects of growth, apoptosis, and cellular signaling. Although a significant number of enzymatic regulators of the sphingolipid pathway have been described in detail, many remained poorly characterized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor family member IV (PAQR4) as a Golgi-localized ceramidase. PAQR4 was approximately 5-fold upregulated in breast cancer compared with matched control tissue and its overexpression correlated with disease-specific survival rates in breast cancer. Induction of PAQR4 in breast tumors was found to be subtype-independent and correlated with increased ceramidase activity. These findings establish PAQR4 as Golgi-localized ceramidase required for cellular growth in breast cancer. SIGNIFICANCE: Induction of and cellular dependency on de novo sphingolipid synthesis via PAQR4 highlights a central vulnerability in breast cancer that may serve as a viable therapeutic target.

Role of Ceramidases in Sphingolipid Metabolism and Human Diseases.

Human pathologies such as Alzheimer's disease, type 2 diabetes-induced insulin resistance, cancer, and cardiovascular diseases have altered lipid homeostasis. Among these imbalanced lipids, the bioactive sphingolipids ceramide and sphingosine-1 phosphate (S1P) are pivotal in the pathophysiology of these diseases. Several enzymes within the sphingolipid pathway contribute to the homeostasis of ceramide and S1P. Ceramidase is key in the degradation of ceramide into sphingosine and free fatty acids. In humans, five different ceramidases are known-acid ceramidase, neutral ceramidase, and alkaline ceramidase 1, 2, and 3-which are encoded by five different genes (ASAH1, ASAH2, ACER1, ACER2, and ACER3, respectively). Notably, the neutral ceramidase N-acylsphingosine amidohydrolase 2 (ASAH2) shows considerable differences between humans and animals in terms of tissue expression levels. Besides, the subcellular localization of ASAH2 remains controversial. In this review, we sum up the results obtained for identifying gene divergence, structure, subcellular localization, and manipulating factors and address the role of ASAH2 along with other ceramidases in human diseases.

Chemotherapy selection pressure alters sphingolipid composition and mitochondrial bioenergetics in resistant HL-60 cells.

The combination of daunorubicin (dnr) and cytarabine (Ara-C) is a cornerstone of treatment for acute myelogenous leukemia (AML); resistance to these drugs is a major cause of treatment failure. Ceramide, a sphingolipid (SL), plays a critical role in cancer cell apoptosis in response to chemotherapy. Here, we investigated the effects of chemotherapy selection pressure with Ara-C and dnr on SL composition and enzyme activity in the AML cell line HL-60. Resistant cells, those selected for growth in Ara-C- and dnr-containing medium (HL-60/Ara-C and HL-60/dnr, respectively), demonstrated upregulated expression and activity of glucosylceramide synthase, acid ceramidase (AC), and sphingosine kinase 1 (SPHK1); were more resistant to ceramide than parental cells; and displayed sensitivity to inhibitors of SL metabolism. Lipidomic analysis revealed a general ceramide deficit and a profound upswing in levels of sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) in HL-60/dnr cells versus parental and HL-60/Ara-C cells. Both chemotherapy-selected cells also exhibited comprehensive upregulations in mitochondrial biogenesis consistent with heightened reliance on oxidative phosphorylation, a property that was partially reversed by exposure to AC and SPHK1 inhibitors and that supports a role for the phosphorylation system in resistance. In summary, dnr and Ara-C selection pressure induces acute reductions in ceramide levels and large increases in S1P and C1P, concomitant with cell resilience bolstered by enhanced mitochondrial remodeling. Thus, strategic control of ceramide metabolism and further research to define mitochondrial perturbations that accompany the drug-resistant phenotype offer new opportunities for developing therapies that regulate cancer growth.

Probing compartment-specific sphingolipids with targeted bacterial sphingomyelinases and ceramidases.

Sphingolipids contribute to the regulation of cell and tissue homeostasis, and disorders of sphingolipid metabolism lead to diseases such as inflammation, stroke, diabetes, and cancer. Sphingolipid metabolic pathways involve an array of enzymes that reside in specific subcellular organelles, resulting in the formation of many diverse sphingolipids with distinct molecular species based on the diversity of the ceramide (Cer) structure. In order to probe compartment-specific metabolism of sphingolipids in this study, we analyzed the Cer and SM species preferentially produced in the inner plasma membrane (PM), Golgi apparatus, ER, mitochondria, nucleus, and cytoplasm by using compartmentally targeted bacterial SMases and ceramidases. The results showed that the length of the acyl chain of Cer becomes longer according to the progress of Cer from synthesis in the ER to the Golgi apparatus, then to the PM. These findings suggest that each organelle shows different properties of SM-derived Cers consistent with its emerging distinct functions in vitro and in vivo.

Gene cloning of a neutral ceramidase from the sphingolipid metabolic pathway based on transcriptome analysis of Amorphophallus muelleri.

Amorphophallus is a perennial herbaceous plant species mainly distributed in the tropics or subtropics of Asia and Africa. It has been used as a traditional medicine for a long time and now is utilized for the pharmaceutical, chemical and agriculture industries as a valued economic crop. Recently, Amorphophallus has attracted tremendous interest because of its high ceramide content. However, the breeding and genome studies are severely limited by the arduous whole genome sequencing of Amorphophallus. In this study, the transcriptome data of A. muelleri was obtained by utilizing the high-throughput Illumina sequencing platform. Based on this information, the majority of the significant genes involved in the proposed sphingolipid metabolic pathway were identified. Then, the full-length neutral ceramidase cDNA was obtained with the help of its candidate transcripts, which were acquired from the transcriptome data. Furthermore, we demonstrated that this neutral ceramidase was a real ceramidase by eukaryotic expression in the yeast double knockout mutant Δypc1 Δydc1, which lacks the ceramidases-dihydroCDase (YDC1p), phytoCDase (YPC1p). In addition, the biochemical characterization of purified A. muelleri ceramidase (AmCDase) exhibited classical Michaelis-Menten kinetics with an optimal activity ranging from pH 6.5 to 8.0. Based on our knowledge, this study is the first to report the related information of the neutral ceramidase in Amorphophallus. All datasets can provide significant information for related studies, such as gene expression, genetic improvement and application on breeding in Amorphophallus.

Ceramidase critically affects GPVI-dependent platelet activation and thrombus formation.

Platelet aggregation, dense granule secretion and thrombus formation are dependent on sphingolipids like ceramide and sphingosine as well as sphingosine-1 phosphate. Sphingosine/ceramide metabolism involves ceramide synthases and ceramidases. However, the role of ceramide synthase and ceramidase in the regulation of platelet function remained ill-defined. The present study determined transmission light aggregometry, employed luciferase based ATP release measurements and studied in vitro thrombus formation under high arterial shear rates in order to define the impact of pharmacological inhibition of serine palmitoyltransferase, ceramide synthase and ceramidase on platelet function. As a result, inhibition of ceramidase significantly blunted collagen related peptide (CRP) induced glyocoprotein VI (GPVI)-dependent platelet aggregation, ATP release and thrombus formation on a collagen-coated surface under shear rates of 1700-sec. Defective platelet aggregation after ceramidase inhibition could partially be overcome by exogenous sphingosine treatment reflecting a pivotal role of ceramidase-derived sphingosine in platelet function. Inhibition of serine palmitoyltransferase and ceramide synthase did not significantly modify GPVI-dependent platelet activation. In conclusion, the present study unraveled ceramidase as a crucial player in sphingosine-induced platelet activation following GPVI-dependent signaling.

Immunologic and endocrine functions of adipose tissue: implications for kidney disease.

Excess adiposity can induce adverse sequelae in multiple cell types and organ systems. The transition from the lean to the obese state is characterized by fundamental cellular changes at the level of the adipocyte. These changes affect the local microenvironment within the respective adipose tissue but can also affect nonadipose systems. Adipocytes within fat pads respond to chronic nutrient excess through hyperplasia or hypertrophy, which can differentially affect interorgan crosstalk between various adipose depots and other organs. This crosstalk is dependent on the unique ability of the adipocyte to coordinate metabolic adjustments throughout the body and to integrate responses to maintain metabolic homeostasis. These actions occur through the release of free fatty acids and metabolites during times of energy need - a process that is altered in the obese state. In addition, adipocytes release a wide array of signalling molecules, such as sphingolipids, as well as inflammatory and hormonal factors (adipokines) that are critical for interorgan crosstalk. The interactions of adipose tissue with the kidney - referred to as the adipo-renal axis - are important for normal kidney function as well as the response of the kidney to injury. Here, we discuss the mechanistic basis of this interorgan crosstalk, which clearly has great therapeutic potential given the increasing rates of chronic kidney disease secondary to obesity and type 2 diabetes mellitus.

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, Crassostrea virginica.

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2-3.3. This enzyme prefers to hydrolyze the acidic GSLs, II3SO3LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4-4.5. Since oyster ceramidase greatly prefers ceramides with C6 to C8 fatty acids, C6-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C16 and C18 fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.

[Detection of effective treatment of amnestic mild cognitive impairement with cereton by testing of lipids markers]. / Issledovanie éffektivnosti tseretona pri miagkom kognitivnom snizhenii amnesticheskogo tipa na osnove testirovaniia markerov lipidnoi prirody.

AIM: Determination of effectivity and safety of Cereton (Choline alfoscerate, production by Sotex) 1200 mg/day in the treatment of cognitive functioning disorders in patients with amnestic mild cognitive impairment (aMCI) and determining its influence in the process (after a 3 month course of taking the drug) and 3 months after the end of treatment of aMCI on the change in the content of phosphatidylcholine, sphingomyelin, ceramide-metabolite sphingolipids and the activity of genes controlling the synthesis of enzymes, which control ithe metabolism of sphingomyelin and ceramide (sphingomyelinase and ceramidase). MATERIAL AND METHODS: The study involved a group of elderly patients (20 people), consisting of 14 women and 6 men, aged 51 to 82 years (mean age 70.3±9.1 years). The patients' condition met the criteria for diagnosing aMCI syndrome. Analysis of phosphatidylcholine, sphingomyelin and ceramide in the blood plasma of patients was carried out by thin layer chromatography, expression of sphingomyelinase and ceramidase genes by RtPCR. RESULTS AND CONCLUSION: A sharp increase in the content of phosphatidylcholine and ceramide, the product of sphingomyelin hydrolysis, was detected. Expression of genes (acidic sphingomyelinase and ceramidase), controlling the metabolism of ceramide, is significantly reduced in the majority of patients in the treatment with ceretone. An increase in the level of phosphatidylcholine and a decrease in the expression level of the ceramide metabolism genes during treatment with ceretone and other drugs that affect the metabolism of phosphatidylchodine and sphingolipids can be used as markers of the effectiveness of therapy.

Inducible overexpression of adiponectin receptors highlight the roles of adiponectin-induced ceramidase signaling in lipid and glucose homeostasis.

OBJECTIVE: Adiponectin and the signaling induced by its cognate receptors, AdipoR1 and AdipoR2, have garnered attention for their ability to promote insulin sensitivity and oppose steatosis. Activation of these receptors promotes the deacylation of ceramide, a lipid metabolite that appears to play a causal role in impairing insulin signaling. METHODS: Here, we have developed transgenic mice that overexpress AdipoR1 or AdipoR2 under the inducible control of a tetracycline response element. These represent the first inducible genetic models that acutely manipulate adiponectin receptor signaling in adult mouse tissues, which allows us to directly assess AdipoR signaling on glucose and lipid metabolism. RESULTS: Overexpression of either adiponectin receptor isoform in the adipocyte or hepatocyte is sufficient to enhance ceramidase activity, whole body glucose metabolism, and hepatic insulin sensitivity, while opposing hepatic steatosis. Importantly, metabolic improvements fail to occur in an adiponectin knockout background. When challenged with a leptin-deficient genetic model of type 2 diabetes, AdipoR2 expression in adipose or liver is sufficient to reverse hyperglycemia and glucose intolerance. CONCLUSION: These observations reveal that adiponectin is critical for AdipoR-induced ceramidase activation which enhances hepatic glucose and lipid metabolism via rapidly acting "cross-talk" between liver and adipose tissue sphingolipids.