Ceramide Signaling Research Articles

Long-term exposure to triclocarban induces splenic injuries in mice: Insights from spatial metabolomics and lipidomics

Triclocarban (TCC) is a widely used antimicrobial agent and known endocrine-disrupting chemical found in various products. While its potential toxicities on endocrine-related organs have been highlighted in previous studies, the effects of TCC on non-endocrine organs, particularly the spleen, remain largely unknown. Here, we employed a novel approach combining long-term TCC exposure in a mouse model with spatial metabolomics and lipidomics to investigate the effects of TCC on the spleen. Our results showed that TCC exposure significantly altered the splenic organ weight and coefficient and induced obvious pathological alterations. Omic analysis revealed that TCC exposure disrupted the splenic homeostasis, as indicated by the upregulation of glutathione metabolism, ceramide-to-sphingomyelin signaling and biosynthesis of glycerophospholipids. Notably, the data of mass spectrometry imaging (MSI) revealed that TCC accumulated in the red pulp of the mouse spleen, while its metabolites concentrated in the white pulp. Further MSI analyses identified region-specific metabolic disruptions, including upregulated ceramide signaling in the red pulp, indicating localized inflammation, and upregulated glutathione metabolism throughout the spleen, suggesting widespread oxidative damage. Our findings provide crucial insights into the spatial distribution and biochemical impact of TCC on mice spleens, highlighting the potential risks of long-term TCC exposure to immune function.

Abstract A066: nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer

Abstract The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. We investigated the effects of eliminating nSMase2 on early and advanced PDA, including its influence on the microenvironment. Employing the KPC mouse model of pancreatic cancer, we demonstrate that pancreatic epithelial nSMase2 ablation reduces neoplasia and promotes a PDA subtype switch from aggressive basal-like to classical. nSMase2 elimination prolongs survival of KPC mice, hinders vasculature development, and fosters a robust immune response. nSMase2 loss leads to recruitment of cytotoxic T cells, N1-like neutrophils, and abundant infiltration of anti-tumorigenic macrophages in the pancreatic preneoplastic microenvironment. Mechanistically, we demonstrate that nSMase2-expressing PDA cell small extracellular vesicles (sEVs) reduce survival of KPC mice; PDA cell sEVs generated independently of nSMase2 prolong survival of KPC mice and reprogram macrophages to a proinflammatory phenotype. Collectively, our study highlights previously unappreciated opposing roles for exosomes, based on biogenesis pathway, during PDA progression. Citation Format: Audrey M Hendley, Sudipta Ashe, Atsushi Urano, Martin Ng, Tuan A Phu, Xianlu Laura Peng, Changfei Luan, Anna-Marie Finger, Gun Ho Jang, Natanya R Kerper, David I Berrios, David Jin, Jonghyun Lee, Irene R Riahi, Oghenekevwe M Gbenedio, Christina Chung, Jeroen P Roose, Jen Jen Yeh, Steven Gallinger, Andrew V Biankin, Grainne M O’Kane, Vasilis Ntranos, David K Chang, David W Dawson, Grace E Kim, Valerie M Weaver, Robert L Raffai, Matthias Hebrok. nSMase2-mediated exosome secretion shapes the tumor microenvironment to immunologically support pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A066.

Palmitic acid induces β-cell ferroptosis by activating ceramide signaling pathway

Individuals with type 2 diabetes mellitus frequently display heightened levels of palmitic acid (PA) in their serum, which may lead to β-cell damage. The involvement of ferroptosis, a form of oxidative cell death in lipotoxic β-cell injury remains uncertain. Here, we have shown that PA induces intracellular lipid peroxidation, increases intracellular Fe2+ content and decreases intracellular glutathione peroxidase 4 (GPX4) expression. Furthermore, PA causes distinct changes in pancreatic islets and INS-1 cells, such as mitochondrial atrophy and increased membrane density. Furthermore, the presence of the ferroptosis inhibitor has a significant mitigating effect on PA-induced β-cell damage. Mechanistically, PA increased ceramide content and c-Jun N-terminal kinase (JNK) phosphorylation. The ceramide synthase inhibitor effectively attenuated PA-induced β-cell damage and GPX4/Fe2+ abnormalities, while inhibiting JNK phosphorylation. Additionally, the JNK inhibitor SP600125 improved PA-induced cell damage. In conclusion, by promoting ceramide synthesis, PA inhibited GPX4 expression and increased intracellular Fe2+ to induce β-cell ferroptosis. Moreover, JNK may be a downstream mechanism of ceramide-triggered lipotoxic ferroptosis in β-cells.

Study on the mechanism of modified Gegen Qinlian decoction in regulating the intestinal flora-bile acid-TGR5 axis for the treatment of type 2 diabetes mellitus based on macro genome sequencing and targeted metabonomics integration

BackgroundCurrently, there are many drugs available for the treatment of type 2 diabetes mellitus (T2DM), but most of them cause various side effects due to the need for long-term use. As a traditional Chinese medicine, Gegen Qinlian Decoction (GQD) has shown good efficacy and low side effects in the treatment of T2DM in both clinical and basic research. Based on relevant traditional Chinese medicine theories, dried ginger is innovatively added the formula of traditional GQD to create a modified GQD. This modification reduces the side effects of traditional GQD while exerting its therapeutic effect on T2DM. Previous studies have found that the modified GQD can regulate endoplasmic reticulum stress in the liver, inhibit hepatic gluconeogenesis, protect pancreatic islet β cells, and control blood sugar levels by inhibiting the FXR/neuronal ceramide signaling pathway. GQD can also regulate the intestinal microbiota to achieve therapeutic and protective effects in various gastrointestinal diseases. However, there is no research exploring whether the modified GQD achieves its therapeutic mechanism for T2DM by regulating the intestinal microbiota. PurposeTo explore the mechanism of modified GQD in the treatment of T2DM based on multi-omics, focusing on its effect on the “intestinal flora bile acid TGR5′' axis. MethodsThe T2DM model was established using db/db mice, which were randomly divided into a model group, metformin group, high-dose GQD group, medium-dose GQD group, low-dose GQD group, while m/m mice were used as blank control. The drug intervention lasted for 12 weeks, during which the general conditions, oral glucose tolerance (OGT), blood glucose, and lipid-related indexes were recorded. Additionally, the fasting insulin (FINS), c-peptide, GLP-1 in serum, and cAMP in the ileum were measured by ELISA assay. Furthermore, the composition, abundance, and function of the intestinal microbiota were determined by macro genome sequencing, while bile acid was detected by targeted metabonomics. For histological evaluation, HE staining was used to observe the pathological changes of the ileum and pancreas, and the ultrastructure of the ileum and pancreas was observed by transmission electron microscopy. Apoptosis in the ileum tissue was detected by Tunel staining. Moreover, the mRNA and protein expressions of TGR5, PKA, CREB, PC1/3, GLP-1, and their phosphorylation levels in the ileum were detected by qPCR, immunohistochemistry, and Western blot; The expression of INS in the pancreas was also evaluated using immunohistochemistry. Finally, double immunofluorescence staining was used to detect the co-localization expression of TGR5 and GLP-1, NeuroD1, and GLP-1 in the ileum. ResultsThe modified GQD was found to significantly reduce blood glucose, improve oral glucose tolerance, and blood lipid levels, as well as alleviate the injury of the ileum and pancreas in T2DM mice. Furthermore, modified GQD was found to effectively regulate intestinal flora, improve bile acid metabolism, activate the TRG5/cAMP/PKA/CREB signal pathway, and stimulate GLP-1 secretion. ConclusionGQD can regulate the “intestinal flora-bile acid-TGR5” axis and has a therapeutic effect on T2DM in mice.

Integrated metabolomics analysis reveals mechanistic insights into variability in blood pressure response to thiazide diuretics and beta blockers.

Hypertensive patients with a higher proportion of genetic West African ancestry (%GWAA) have better blood pressure (BP) response to thiazide diuretics (TDs) and worse response to β-blockers (BBs) than those with lower %GWAA, associated with their lower plasma renin activity (PRA). TDs and BBs are suggested to reduce BP in the long term through vasodilation via incompletely understood mechanisms. This study aimed at identifying pathways underlying ancestral differences in PRA, which might reflect pathways underlying BP-lowering mechanisms of TDs and BBs. Among hypertensive participants enrolled in the Pharmacogenomics Evaluation of Antihypertensive Responses (PEAR) and PEAR-2 trials, we previously identified 8 metabolites associated with baseline PRA and 4 metabolic clusters (including 39 metabolites) that are different between those with GWAA <45% versus ≥45%. In the current study, using Ingenuity Pathway Analysis (IPA), we integrated these signals. Three overlapping metabolic signals within three significantly enriched pathways were identified as associated with both PRA and %GWAA: ceramide signaling, sphingosine 1- phosphate signaling, and endothelial nitric oxide synthase signaling. Literature indicates that the identified pathways are involved in the regulation of the Rho kinase cascade, production of the vasoactive agents nitric oxide, prostacyclin, thromboxane A2, and endothelin 1; the pathways proposed to underlie TD- and BB-induced vasodilatation. These findings may improve our understanding of the BP-lowering mechanisms of TDs and BBs. This might provide a possible step forward in personalizing antihypertensive therapy by identifying patients expected to have robust BP-lowering effects from these drugs.

Lysosomal Regulation of Gasdermin D Pore Formation in Podocytes Determined by Acid Sphingomyelinase

Recent studies have shown that gasdermin D (GSDMD) pore may be involved in inflammasome product release and pyroptosis in podocytes. Nevertheless, the molecular mechanism by which GSDMD pore formation is regulated in these cells remains poorly understood. Given the important role of acid sphingomyelinase (ASM)-ceramide signaling pathway in glomerular inflammation and sclerosis under pathological conditions, the present study tested whether ASM controls lysosome-dependent autophagic degradation of GSDMD-NT and GSDMD pore repair to determine GSDMD pore formation in podocytes. WT/WT, ASM knockout (KO), and ASM overexpression (OE) podocytes were isolated from wild type, Smpd1 (gene code of ASM) gene KO, and podocyte-specific Smpd1 gene OE mice for experiments. We first demonstrated that palmitic acid (PA) as an obesity-related danger factor induced NLRP3 inflammasome activation, GSDMD pore formation on plasma membrane, and IL-1β secretion in podocytes, which was blocked by Smpd1 gene KO but exaggerated by Smpd1 gene OE. Also, it was found that PA-induced pyroptosis was prevented by ASM KO but amplified by ASM OE in podocytes. By confocal microscopy and super-resolution microscopy, we observed that PA treatment enhanced the formation of autophagosomes containing GSDMD-NT but inhibited lysosome-autophagosome interaction in podocytes. Also, FasL-induced lysosome fusion to plasma membrane was reduced by PA. All these pathological changes were remarkably attenuated by ASM KO or amitriptyline but significantly augmented by ASM OE. To further dissect the molecular mechanism by which ASM determines GSDMD pore formation, we examined whether ASM controls lysosomal TRPML1 channel-mediated Ca2+ release to regulate lysosome traffcking and associated lysosomal functions. By GCaMP3 Ca2+ imaging, we found that PA treatment significantly inhibited TRPML1 channel activity in podocytes, which was exaggerated by ASM OE. Correspondingly, PA-induced reduction of lysosome traffcking was more remarkable in ASM OE podocytes compared to WT/WT podocytes. Moreover, PA-induced decrease in lysosome-autophagosome interaction and increase in GSDMD pore formation in podocytes were hindered by ML-SA5 (TRPML1 channel agonist) but enhanced by ML-SI1 (TRPML1 channel inhibitor). In summary, our findings suggest that ASM plays a crucial role in lysosomal regulation of GSDMD pore formation via control of TRPML1 channel activity and lysosome traffcking in podocytes. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Contribution of Gasdermin D Pore Formation to NLRP3 Inflammasome Product Secretion and Pyroptosis in Podocytes during Obesity-Induced Glomerulopathy

NLRP3 inflammasome activation in podocytes has been reported to play an important role in the development of glomerular inflammation and sclerosis during obesity. Although, the molecular mechanism mediating the secretion of NLRP3 inflammasome products to induce local inflammatory response remains poorly understood. Given that gasdermin D (GSDMD) has been found to form oligomeric pores on plasma membrane for release of inflammasome product and execution of pyroptosis, the present study tested whether GSDMD pore contributes to NLRP3 inflammasome product secretion and pyroptosis in podocytes and thereby initiates glomerular inflammation and sclerosis in obesity-induced glomerulopathy (ORG). By Western blot analysis, we demonstrated that palmitic acid (PA) as an obesity-related danger factor dose-dependently stimulated the reduction of total GSDMD and elevation of cleaved GSDMD (GSDMD-NT) in podocytes. In vivo, glomerular decrease in total GSDMD and increase in GSDMD-NT were remarkable in wild type (WT/WT) mice on high-fat diet (HFD) compared to control mice. Urinary excretion of NLRP3 inflammasome products was also significantly increased by HFD treatment. Moreover, we found that pre-treatment with amitriptyline or MCC950 blocked NLRP3 inflammasome activation as well as GSDMD pore formation on plasma membrane of podocytes, while disulfiram prevented GSDMD pore formation without affecting NLRP3 inflammasome activation. Since acid sphingomyelinase (ASM)-ceramide signaling pathway plays a vital role in the development of ORG, we examined whether ASM activity determines GSDMD pore formation in podocytes. By atom force microscopy, a 3D topography map of a single podocyte was generated, and pore-like structures were observed in the membrane of PA-treated WT/WT podocytes. Podocytes lacking Smpd1 (gene code of ASM) gene did not show pore formation, but Smpd1 gene overexpression amplified PA-induced pore formation in podocytes. Moreover, it was found that HFD-induced GSDMD cleavage and pyroptosis in glomeruli were blocked by Smpd1 gene deletion in Smpd1−/− mice. On the contrary, podocyte-specific Smpd1 gene overexpression amplified GSDMD-NT production and pyroptosis in glomeruli. Correspondingly, HFD-induced podocyte foot process effacement and glomerular sclerosis in mice were inhibited by Smpd1 gene knockout but exaggerated by podocyte-specific Smpd1 gene overexpression. Taken together, our findings suggest that ASM-ceramide signaling pathway may control GSDMD pore formation in podocytes to determine glomerular inflammation and sclerosis in ORG. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 2846: Investigation of mechanisms that result in health disparity for Hispanic/Latino patients with B acute lymphoblastic leukemia

Abstract Although social determinants of health have a large impact on health disparity, biologic vulnerabilities - particularly in cancers that impact at-risk populations are important. Amongst children with Acute Lymphoblastic Leukemia (ALL), the Hispanic/Latino (H/L) population is vulnerable given since the risk of developing ALL is 1.2-1.75 that of Non-Hispanic White children. After corrections for socioeconomic impacts, the mortality for H/L children is 40% higher than NHW. H/L children have a higher rate of high-risk genetic variations: 2-fold rate of heterozygous IKZF1 deletion (IKZF1-del) and a 4-fold rate of CRLF2 translocation. IKZF1 deletion and CRLF2 translocations are associated increased risk of relapse and chemotherapy resistance in B ALL. IKZF1 is an important transcriptional factor and epigenetic regulator that affects global gene transcription in hematopoietic cells. Simultaneous IKZF1-del and CRLF2 translocation represented 94% of H/L children with CRLF2 translocation. Although only 49% of all H/L patients with IKZF1-del had a CRLF2 translocation. Transcriptomics were analyzed for 42 H/L and 18 NHW patient samples using RNAseq. Gene expression from H/L patients was compared to expression in NHW patients and differentially expressed genes were analyzed. Gene set enrichment analysis (GSEA) revealed downregulation of processes responsible for genome maintenance including DNA replication, DNA templated transcription elongation, double strand break repair, and recombinational repair. Ingenuity pathway analysis (IPA) revealed upregulated pathways in stress related translation, histone deacetylases, interferon, and FLT3 signaling compared to NWH. H/L specimens resulted in decreased expression for FGF pathways, ceramide, and FAK signaling. We performed the same analysis in H/L and NHW with wildtype IKZF1 to compare these two population without high risk structural variants. GSEA analysis showed downregulated Intra S DNA Damage checkpoint signaling, DNA replication checkpoint signaling, glutamine amino acid synthesis, and aspartate amino acid synthesis. IPA showed upregulated interferon, histone deacetylases, TREM1 signaling, and Tec Kinase signaling. IPA showed downregulated Ephrin and CDK5 signaling. DNA methylation patterns were compared between 8 H/L and 8 NWH B ALL samples which showed differential methylation patterns affecting multiple cancer relevant genes.In conclusion, these data represent biologic vulnerability in H/L patients with B ALL in comparison to NWH regardless of high-risk genetic variants. H/L B-ALL shows deregulated maintenance of the genome via replication and repair pathways as well as potential vulnerabilities related to regulation of glutamate/aspartate synthesis. These findings give insight into potential mechanisms that can explain increased frequency of high-risk genetic variants in H/L B ALL. Citation Format: Joseph W. Schramm, Bing He, Yali Ding, Chingakham Singh, Daniel Bogush, Katarina Dovat, Diwakar Bastihalli Tukaramrao, Riya Bhalodia, Sinisa Dovat. Investigation of mechanisms that result in health disparity for Hispanic/Latino patients with B acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2846.

Abstract A046: nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma

Abstract The pleiotropic roles of nSMase2-generated ceramide include regulation of intracellular ceramide signaling and exosome biogenesis. Packaging of specific cellular constituents, such as prion protein and specific miRNAs involved in cancer progression, into exosomes are dependent on nSMase2. Employing the KPC mouse model of pancreatic ductal adenocarcinoma (PDA), we demonstrate that nSMase2 regulates exosome secretion and in vivo growth of PDA cells. PDA cell exosomes generated through nSMase2 accelerate PDA progression, and PDA cell exosomes generated independently of nSMase2 are anti-tumorigenic during PDA progression. Homozygous loss of Smpd3 (gene which encodes nSMase2) in KPC mouse pancreatic epithelial cells during development extends survival of KPC mice. KPC; Smpd3f/f mice display less PanIN and tumor burden compared to KPC; Smpd3wt/wt controls. In addition, nSMase2 loss in KPC PDA cells promoted a PDA subtype switch from aggressive basal-like to classical. RNA-sequencing of PDA cell lines generated from pancreatic tumors of end-stage KPC; Smpd3wt/wt, KPC; Smpd3f/wt, and KPC; Smpd3f/f mice demonstrated that nSMase2-generated ceramide is a key regulator of intracellular pathways regulating tumor microenvironment dynamics in PDA cells including fibrosis and integrins. We observed a decrease in extracellular matrix collagen abundance and fewer activated stellate cells and fibroblasts in KPC; Smpd3f/f compared to control KPC; Smpd3wt/wt mouse pancreata. Exosomes isolated from KPC; Smpd3wt/wt PDA cell lines significantly stimulate proliferation of primary murine stellate cells in vitro, while exosomes isolated from KPC; Smpd3f/f PDA cell lines do not. Abrogation of Smpd3 expression also affected immune cell infiltration, as demonstrated by a significant increase in iNOS+ F4/80+ double positive proinflammatory macrophages in KPC; Smpd3f/f pancreata when compared to KPC; Smpd3wt/wt pancreata. Lipidomics analysis of epithelial cell lines generated from end-stage pancreatic tumors of KPC; Smpd3f/f and KPC; Smpd3wt/wt mice demonstrated an alteration in hundreds of lipid species including ceramides, triacylglycerides, sphingomyelins, and phosphatidylcholines. Proteomics and small RNA-seq analysis of exosomes secreted from KPC; Smpd3wt/wt and KPC; Smpd3f/f PDA cell lines demonstrated significant alteration of &amp;gt;700 proteins and 12 miRNAs. Our patient data demonstrate that low nSMase2 expression in treatment naïve primary resected pancreatic tumors significantly correlates with fewer CD31+ endothelial cells. In parallel, loss of Smpd3 resulted in a reduction in CD31+ endothelial cells in pancreatic tumors of KPC; Smpd3f/f mice when compared to KPC; Smpd3wt/wt controls. Thus, nSMase2 is a critical mediator of angiogenesis during PDA progression, which is integral to chemotherapeutic response. Collectively, our data show that nSMase2-generated exosomes promote PDA tumorigenesis, specifically proliferation of stellate cells, which may in turn induce a fibrotic tumor microenvironment that affects vasculature development. Citation Format: Audrey M. Hendley, Atsushi Urano, Xianlu L. Peng, Sudipta Ashe, Tuan A. Phu, Martin Ng, Natanya R. Kerper, David I. Berrios, Jonghyun Lee, David Jin, Gun H. Jang, Oghenekevwe M. Gbenedio, Jeroen P. Roose, Jen J. Yeh, Steven Gallinger, Andrew V. Biankin, Vasilis Ntranos, David K. Chang, David W. Dawson, Grace E. Kim, Valerie M. Weaver, Robert L. Raffai, Matthias Hebrok. nSMase2-generated ceramide promotes PDA aggression through exosome reprogramming of the stroma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A046.

Biological function, topology, and quantification of plasma membrane Ceramide

Over the past 30 years, a growing body of evidence has revealed the regulatory role of the lipid ceramide in various cellular functions. The structural diversity of ceramide, resulting in numerous species, and its distinct distribution within subcellular compartments may account for its wide range of functions. However, our ability to study the potential role of ceramide in specific subcellular membranes has been limited. Several works have shown mitochondrial, Golgi, and plasma membrane ceramide to mediate signaling pathways independently. These results have started to shift the focus on ceramide signaling research toward specific membrane pools. Nonetheless, the challenge arises from the substantial intracellular ceramide content, hindering efforts to quantify its presence in particular membranes. Recently, we have developed the first method capable of detecting and quantifying ceramide in the plasma membrane, leading to unexpected results such as detecting different pools of ceramide responding to drug concentration or time. This review summarizes the historical context that defined the idea of pools of ceramide, the studies on plasma membrane ceramide as a bioactive entity, and the tools available for its study, especially the new method to detect and, for the first time, quantify plasma membrane ceramide. We believe this method will open new avenues for researching sphingolipid signaling and metabolism.

CerS2 ko in vivo reduces pro-inflammatory ceramides and improves doxorubicin mediated cardiotoxicity

Abstract Background Ceramides and their role in various signaling pathways are believed to regulate distinct cellular effects. Accumulation of ceramides seems to be associated with an inflammatory phenotype negatively affecting cardiomyocyte vitality and mitochondrial homeostasis. Aim The purpose of our study is to identify underlying ceramide signaling pathways and characterize downstream effects on cell vitality of cardiomyocytes. Methods A CerS2 ko mouse model was generated and the animals were treated with 7 cumulative doses of 3 mg/kg BW Doxorubicin (Dox). Subsequently, hearts were extracted and analyzed, including mRNA (qPCR) and protein expression (WB) and ceramide level (mass spectrometry). Results The CerS2 ko was stable and Dox injection did not increase CerS2 protein expression. Mass spectrometry revealed an increase of long chain ceramides (C≤20) with the highest peak for C18:0 (5.08 ± 0.21, p&amp;lt;0.001) but a decrease of the pro- inflammatory very long chain ceramides C24:0 (0.65 ± 0.20, p = 0.02) and C24:1 (0.23 ± 0.22, p&amp;lt;0.001). qPCR showed a decrease of TNFα (0.18 ± 0.49, p&amp;lt;0.001), IL-1β (0.43 ± 0.27, p&amp;lt;0.001) and BNP (0.28 ± 0.53, p&amp;lt;0.001). Furthermore, mitochondrial homeostasis improved by a reduction of the mitochondria fission related gene Mitochondrial fission factor (Mff) (0.74 ± 0.23, p = 0.007). Conclusion CerS2 ko and subsequent ceramide reduction improves cardiomyocyte vitality on a cellular level by a reduction of inflammation and an improvement of mitochondrial function. Thus, regulation of ceramide levels through control of CerS2 activity might represent a novel therapeutic option for the control of cardio toxicity.

Use of multi-walled carbon nanotubes (MWCNTs) stabilized in Arabic gum colloidal solution to induce genotoxicity and apoptosis of human breast and lung cancer cell lines

Introduction: Increased mortality owing to breast and lung cancer is an urgent global medical issue. Nanotherapy is one of the most widely used and recent procedures for combating cancer. Therefore, this study aimed to assess the beneficial effects of multi-walled carbon nanotubes (MWCNTs) to precisely target breast (MCF-7) and lung cancer (A549) cell lines. Multi-walled carbon nanomaterials are considered as a typical biocompatible nanomaterial that can be used in drug delivery without causing side effects. On the contrary, the metallic nanoparticles, even that of noble metals, are reported to have side effects that are still unavoidable.Methods: The MWCNTs were purified and stabilized using Arabic gum in an aqueous solution under ultrasonic radiation. Scanning and transmission electron microscopy (SEM and TEM, respectively) were employed to characterize the morphology of the MWCNTs, and elemental analysis was performed using SEM to determine their chemical composition.Results: The investigated MWCNTs had an average diameter of 10–12 nm and were mainly composed of 99.4% carbon and other trace elements. The Arabic gum emulsifying agent was found a powerful stabilizing agent for the investigated MWCNTs in aqueous solutions. The cytotoxic effect of the MWCNT/ Arabic gum colloidal solution on MCF-7 and A549 was evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, and the expression of apoptosis-related genes was assessed using quantitative real-time polymerase chain reaction and a human apoptosis signaling antibody array. This analysis aimed to clarify additional pathways involved, including those mediated by interleukin-1, tumor necrosis factor-α, and ceramide signaling pathways enrichments. Concerning the inhibition of cell growth, the efficacy of MWCNT/Arabic gum IC50 of MCF-7 (48.23 μg/mL) was higher than IC50 of A549 (100.1 μg/mL). MWCNT/Arabic gum enhanced the mRNA expression of apoptosis-associated genes encoding P53, Bax, and Caspases (9, 8, and 3). Upregulation of the apoptotic signaling pathway in the MCF-7 cell lysates was observed before and after exposure to the MWCNT/Arabic gum colloidal solution.Conclusion: The physicochemical properties of MWCNT/Arabic gum exhibited remarkable apoptotic activity in breast and lung cancer cell lines. However, further in vivo investigations are recommended to clarify the other molecular aspects related to the use of MWCNT/Arabic gum.

Study of Ceramide-Flavone Analogs Showing Self-Fluorescence and Anti-Proliferation Activities.

Many current anti-cancer drugs used to treat breast cancer mediate tumor cell death through the induction of apoptosis. Cancer cells, however, often acquire multidrug-resistance following prolonged exposure to chemotherapeutics. Consequently, molecular pathways involved in tumor cell proliferation have become potential targets for pharmacological intervention. Ceramides are tumor suppressor lipids naturally found in the cell membrane, and are central molecules in the sphingolipid signalling pathway. Our lab has targeted the ceramide signaling pathway for potential pharmacological intervention in the treatment of breast cancer. Previously, we have shown that certain ceramide analogs have therapeutic potential in the treatment of chemo-sensitive and multidrug-resistant breast cancers. Using the most active analog from our previous studies as the lead compound, new analogs containing a flavone moiety were designed and synthesized. In general, flavone derivatives often show interesting pharmacological properties, and compounds based on these molecules have been found useful in many different therapeutic areas including anti-tumor, anti-coagulants, and anti-HIV therapy. Synthesis and biological evaluation of five new flavonoid ceramide analogs are reported here. These compounds were also shown to be self-fluorescent, which can be useful when investigating their distribution and action in cancer cells. Four out of the five flavone ceramide analogs in this study showed significant anti-proliferation activities in the three cell lines studied, MDA-MB-232, MCF-7, and MCF-7TN-R; some showing varying degrees of selectivity. The mechanisms involved in cell proliferation inhibition are complicated and further studies are needed.

Molecular tracking of insulin resistance and inflammation development on visceral adipose tissue.

Visceral adipose tissue (VAT) is one of the most important sources of proinflammatory molecules in obese people and it conditions the appearance of insulin resistance and diabetes. Thus, understanding the synergies between adipocytes and VAT-resident immune cells is essential for the treatment of insulin resistance and diabetes. We collected information available on databases and specialized literature to construct regulatory networks of VAT resident cells, such as adipocytes, CD4+ T lymphocytes and macrophages. These networks were used to build stochastic models based on Markov chains to visualize phenotypic changes on VAT resident cells under several physiological contexts, including obesity and diabetes mellitus. Stochastic models showed that in lean people, insulin produces inflammation in adipocytes as a homeostatic mechanism to downregulate glucose intake. However, when the VAT tolerance to inflammation is exceeded, adipocytes lose insulin sensitivity according to severity of the inflammatory condition. Molecularly, insulin resistance is initiated by inflammatory pathways and sustained by intracellular ceramide signaling. Furthermore, our data show that insulin resistance potentiates the effector response of immune cells, which suggests its role in the mechanism of nutrient redirection. Finally, our models show that insulin resistance cannot be inhibited by anti-inflammatory therapies alone. Insulin resistance controls adipocyte glucose intake under homeostatic conditions. However, metabolic alterations such as obesity, enhances insulin resistance in adipocytes, redirecting nutrients to immune cells, permanently sustaining local inflammation in the VAT.

Distinct T cell subsets in adipose tissue are associated with obesity.

Adipose tissue inflammation is a driving factor for the development of obesity-associated metabolic disturbances, and a role of adipose tissue T cells in initiating the pro-inflammatory signaling is emerging. However, data on human adipose tissue T cells in obesity are limited, reflected by the lack of phenotypic markers to define tissue-resident T cell subsets. In this study, we performed a deep characterization of T cells in blood and adipose tissue depots using multicolor flow cytometry and RNA sequencing. We identified distinct subsets of T cells associated with obesity expressing the activation markers, CD26 and CCR5, and obesity-specific genes that are potentially engaged in activating pro-inflammatory pathway, including ceramide signaling, autophagy, and IL-6 signaling. These findings increase our knowledge on the heterogeneity of T cells in adipose tissue and on subsets that may play a role in obesity-related pathogenesis.

Long chain ceramides raise the main phase transition of monounsaturated phospholipids to physiological temperature

Little is known about the molecular mechanisms of ceramide-mediated cellular signaling. We examined the effects of palmitoyl ceramide (C16-ceramide) and stearoyl ceramide (C18-ceramide) on the phase behavior of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) using differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS, WAXS). As previously published, the presence of ceramides increased the lamellar gel-to-lamellar liquid crystalline (Lβ–Lα) phase transition temperature of POPC and POPE and decreased the Lα-to-inverted hexagonal (Lα–HII) phase transition temperature of POPE. Interestingly, despite an ~ 30° difference in the main phase transition temperatures of POPC and POPE, the Lβ–Lα phase transition temperatures were very close between POPC/C18-ceramide and POPE/C18-ceramide and were near physiological temperature. A comparison of the results of C16-ceramide in published and our own results with those of C18-ceramide indicates that increase of the carbon chain length of ceramide from 16 to 18 and/or the small difference of ceramide content in the membrane dramatically change the phase transition temperature of POPC and POPE to near physiological temperature. Our results support the idea that ceramide signaling is mediated by the alteration of lipid phase-dependent partitioning of signaling proteins.

Abstract C051: Ceramide signaling regulates PDA aggression through exosome reprogramming of the stroma

Abstract Ceramide is a bioactive lipid signaling molecule that regulates multiple cellular processes influencing pancreatic tumor progression and drug response. The pleiotropic role of ceramide signaling in cancer includes modulation of exosome biogenesis and secretion. Smpd3 encodes an enzyme that generates ceramide through hydrolysis of sphingomyelin. Employing the KPC mouse model of pancreatic cancer, we demonstrated that Smpd3 regulates exosome biogenesis in pancreatic ductal adenocarcinoma (PDA) cells and is pro-tumorigenic during PDA progression. Ablation of Smdp3 in KPC mice significantly extends survival by 19% when compared to KPC; Smpd3wt/wt controls. KPC; Smpd3f/f mice display significantly less PanIN and tumor burden compared to KPC; Smpd3wt/wt controls. Lipidomics analysis of epithelial cell lines generated from end-stage pancreatic tumors of KPC; Smpd3f/f and KPC; Smpd3wt/wt mice demonstrated an alteration in hundreds of lipid species including ceramides, triacylglycerides, sphingomyelins, and phosphatidylcholines. Analysis of RNA-seq data of these epithelial cell lines showed a switching of primary tumors from the predominant more aggressive basal-like subtype seen in KPC; Smpd3wt/wt mice to classical in KPC; Smpd3f/f mice. Pathways analysis of our RNA-seq dataset showed an enrichment for genes involved in cellular mechanics and regulation of the tumor microenvironment. To query if Smpd3-generated exosomes have a direct effect on pancreatic tumor progression, we injected KPC; Smpd3wt/wt and KPC; Smpd3f/f mice with exosomes isolated from KPC; Smpd3f/f and KPC; Smpd3wt/wt PDA cell lines. Injection of exosomes derived from KPC; Smpd3f/f mice significantly extended survival of both Smpd3wt/wt and KPC; Smpd3f/f mice when compared to injection of exosomes isolated from KPC; Smpd3wt/wt mice, suggesting an anti-tumorigenic effect of exosomes isolated from Smpd3-deficient PDA cell lines. We observed a decrease in extracellular matrix collagen abundance and fewer activated stellate cells and fibroblasts in KPC; Smpd3f/f compared to control KPC; Smpd3wt/wt pancreata. Abrogation of Smpd3 expression also affected immune cell infiltration, as demonstrated by a significant increase in iNOS+ F4/80+ double positive macrophages in KPC; Smpd3f/f pancreata when compared to KPC; Smpd3wt/wt pancreata. Loss of Smpd3 resulted in a significant reduction in CD31+ endothelial cells in pancreatic tumors of KPC; Smpd3f/f mice when compared to KPC; Smpd3wt/wt mice, which may influence the ability of chemotherapeutics to enter pancreatic tumors. Our patient data demonstrate that high SMPD3 expression in surgically resected, treatment naive PDA significantly correlated with longer patient survival when patients received adjuvant chemotherapy, more than 95% of which was gemcitabine. Collectively, our data show that ceramide-dependent exosomes promote tumorigenesis, specifically activation of stellate cells and fibroblasts – which may in turn induce a stiff, fibrotic, proinflammatory tumor microenvironment that also impedes vasculature formation. Citation Format: Audrey M. Hendley, Atsushi Urano, Xianlu L. Peng, Sudipta Ashe, Natanya R. Kerper, Tuan A. Phu, Martin Ng, Simone Giacometti, David I. Berrios, Gun H. Jang, Jen J. Yeh, Steven Gallinger, David K. Chang, Andrew V. Biankin, Valerie M. Weaver, Grace E. Kim, David W. Dawson, Robert L. Raffai, Matthias Hebrok. Ceramide signaling regulates PDA aggression through exosome reprogramming of the stroma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C051.

Endothelial Acid Sphingomyelinase Promotes NLRP3 Inflammasome and Neointima Formation During Hypercholesterolemia

The NOD-like receptor pyrin domain 3 (NLRP3) inflammasome is activated during atherogenesis, but how this occurs is unclear. Here, we explored the mechanisms activating and regulating NLRP3 inflammasomes via the acid sphingomyelinase (ASM)-ceramide signaling pathway. As a neointima formation model, partial left carotid ligations were performed on endothelial cell (EC)-specific ASM transgene mice (Smpd1trg/ECcre) and their control littermates (Smpd1trg/WT and WT/WT) fed on the Western diet (WD). We found neointima formation remarkably increased in Smpd1trg/ECcre mice over their control littermates. Next, we observed enhanced colocalization of NLRP3 versus adaptor protein ASC (the adaptor molecule apoptosis-associated speck-like protein containing a CARD) or caspase-1 in the carotid ECs of WD-treated Smpd1trg/ECcre mice but not in their control littermates. In addition, we used membrane raft (MR) marker flotillin-1 and found more aggregation of ASM and ceramide in the intima of Smpd1trg/ECcre mice than their control littermates. Moreover, we demonstrated by in situ dihydroethidium staining, carotid intimal superoxide levels were much higher in WD-treated Smpd1trg/ECcre mice than in their control littermates. Using ECs from Smpd1trg/ECcre and WT/WT mice, we showed ASM overexpression markedly enhanced 7-ketocholesterol (7-Ket)-induced increases in NLRP3 inflammasome formation, accompanied by enhanced caspase-1 activity and elevated interleukin-1β levels. These 7-Ket-induced increases were significantly attenuated by ASM inhibitor amitriptyline. Furthermore, we determined that increased MR clustering with NADPH oxidase subunits to produce superoxide contributes to 7-Ket-induced NLRP3 inflammasome activation via a thioredoxin-interacting protein-mediated controlling mechanism. We conclude that ceramide from ASM plays a critical role in NLRP3 inflammasome activation during hypercholesterolemia via MR redox signaling platforms to produce superoxide, which leads to TXNIP dissociation.

Exercise Promotes Pro-Apoptotic Ceramide Signaling in a Mouse Melanoma Model.

Simple SummaryExercise has been shown to improve the efficacy of chemotherapy against several tumor models using mice through modulating tumor vascular perfusion, immune function, circulating growth factors, hypoxia, and metabolism in tumor cells and their surrounding microenvironment. However, little is known about the effect of exercise on tumor-cell-intrinsic death mechanisms, such as apoptosis. Ceramide is a bioactive lipid that can promote cell death. The strategy of increasing intracellular ceramide has potential as an anticancer treatment for melanoma with dysregulated ceramide metabolism, but there is not yet a clinically relevant method to do so. We found that moderate aerobic exercise increases pro-apoptotic ceramide in melanoma in mice, and activates p53 signaling, promoting tumor cell apoptosis. This finding suggests that exercise may be most effective as an adjuvant therapy to sensitize cancer cells to anticancer treatments in tumors that exhibit downregulated ceramide generation to evade cell death.Ceramides are essential sphingolipids that mediate cell death and survival. Low ceramide content in melanoma is one mechanism of drug resistance. Thus, increasing the ceramide content in tumor cells is likely to increase their sensitivity to cytotoxic therapy. Aerobic exercise has been shown to modulate ceramide metabolism in healthy tissue, but the relationship between exercise and ceramide in tumors has not been evaluated. Here, we demonstrate that aerobic exercise causes tumor cell apoptosis and accumulation of pro-apoptotic ceramides in B16F10 but not BP melanoma models using mice. B16F10 tumor-bearing mice were treated with two weeks of moderate treadmill exercise, or were control, unexercised mice. A reverse-phase protein array was used to identify canonical p53 apoptotic signaling as a key pathway upregulated by exercise, and we demonstrate increased apoptosis in tumors from exercised mice. Consistent with this finding, pro-apoptotic C16-ceramide, and the ceramide generating enzyme ceramide synthase 6 (CerS6), were higher in B16F10 tumors from exercised mice, while pro-survival sphingosine kinase 1 (Sphk1) was lower. These data suggest that exercise contributes to B16F10 tumor cell death, possibly by modulating ceramide metabolism toward a pro-apoptotic ceramide/sphingosine-1-phosphate balance. However, these results are not consistent in BP tumors, demonstrating that exercise can have different effects on tumors of different patient or mouse origin with the same diagnosis. This work indicates that exercise might be most effective as a therapeutic adjuvant with therapies that kill tumor cells in a ceramide-dependent manner.

Inimitable Impacts of Ceramides on Lipid Rafts Formed in Artificial and Natural Cell Membranes

Ceramide is the simplest precursor of sphingolipids and is involved in a variety of biological functions ranging from apoptosis to the immune responses. Although ceramide is a minor constituent of plasma membranes, it drastically increases upon cellular stimulation. However, the mechanistic link between ceramide generation and signal transduction remains unknown. To address this issue, the effect of ceramide on phospholipid membranes has been examined in numerous studies. One of the most remarkable findings of these studies is that ceramide induces the coalescence of membrane domains termed lipid rafts. Thus, it has been hypothesised that ceramide exerts its biological activity through the structural alteration of lipid rafts. In the present article, we first discuss the characteristic hydrogen bond functionality of ceramides. Then, we showed the impact of ceramide on the structures of artificial and cell membranes, including the coalescence of the pre-existing lipid raft into a large patch called a signal platform. Moreover, we proposed a possible structure of the signal platform, in which sphingomyelin/cholesterol-rich and sphingomyelin/ceramide-rich domains coexist. This structure is considered to be beneficial because membrane proteins and their inhibitors are separately compartmentalised in those domains. Considering the fact that ceramide/cholesterol content regulates the miscibility of those two domains in model membranes, the association and dissociation of membrane proteins and their inhibitors might be controlled by the contents of ceramide and cholesterol in the signal platform.