Ceramide Levels Research Articles

Acid sphingomyelinase mediates ferroptosis induced by high glucose via autophagic degradation of GPX4 in type 2 diabetic osteoporosis

BackgroundFerroptosis has been implicated in the pathological process of type 2 diabetic osteoporosis (T2DOP), although the specific underlying mechanisms remain largely unknown. This study aimed to clarify the role and possible mechanism of acid sphingomyelinase (ASM)-mediated osteoblast ferroptosis in T2DOP.MethodsWe treated hFob1.19 cells with normal glucose (NG) and different concentrations of high glucose (HG, 26.25 mM, 35 mM, or 43.75 mM) for 48 h. We then measured cell viability and osteogenic function, quantified ferroptosis and autophagy levels, and measured the levels of ASM and ceramide in the cells. To further investigate the specific mechanism, we examined these indicators by knocking down ASM expression, hydroxychloroquine (HCQ) treatment, or N-acetylcysteine (NAC) treatment. Moreover, a T2DOP rat model was induced and microcomputed tomography was used to observe the bone microstructure. We also evaluated the serum levels of iron metabolism-associated factors, ceramide and lipid peroxidation (LPO) and measured the expression of ASM, LC3 and GPX4 in bone tissues.ResultsHG inhibited the viability and osteogenic function of osteoblasts by inducing ferroptosis in a concentration-dependent manner. Furthermore, the expression of ASM and ceramide and autophagy levels were increased by HG treatment, and these factors were required for the HG-induced reactive oxygen species (ROS) generation and LPO. Similarly, inhibiting intracellular ROS also reduced HG-induced ASM activation and autophagy. ASM-mediated activation of autophagy was crucial for HG-induced degradation of GPX4, and inhibiting ASM improved osteogenic function by decreasing HG-induced autophagy, GPX4 degradation, LPO and subsequent ferroptosis. We also found that inhibiting ASM could alleviated ferroptosis and autophagy and improved osteogenic function in a T2DOP rat model.ConclusionASM-mediated autophagy activation induces osteoblast ferroptosis under HG conditions through the degradation of GPX4, providing a novel mechanistic insight into the treatment and prevention of T2DOP.

Immunometabolic capacities of nutritional fatty acids in regulation of inflammatory bone cell interaction and systemic impact of periodontal infection.

Novel preventive strategies in periodontal disease target the bacterial-induced inflammatory host response to reduce associated tissue destruction. Strategies focus on the modulation of tissue-destroying inflammatory host response, particularly the reduction of inflammation and promotion of resolution. Thereby, nutrition is a potent immunometabolic non-pharmacological intervention. Human studies have demonstrated the benefit of olive oil-containing Mediterranean-style diets (MDs), the main component of which being mono-unsaturated fatty acid (FA) oleic acid (OA (C18:1)). Hence, nutritional OA strengthened the microarchitecture of alveolar trabecular bone and increased circulating pro-resolving lipid mediators following bacterial inoculation with periodontal pathogen Porphyromonas gingivalis, contrary to saturated FA palmitic acid (PA (C16:0)), which is abundant in Western-style diets. Additionally, the generalized distribution of inflammatory pathway mediators can occur in response to bacterial infection and compromise systemic tissue metabolism and bone homeostasis distant from the side of infection. Whether specific FA-enriched nutrition and periodontal inoculation are factors in systemic pathology that can be immune-modulatory targeted through dietary substitution is unknown and of clinical relevance. Normal-weight C57BL/6-mice received OA-or PA-enriched diets (PA-ED, OA-ED, PA/OA-ED) or a normal-standard diet (n=12/group) for 16 weeks and were orally infected with P. gingivalis/placebo to induce periodontal disease. Using histomorphometry and LC-MS/MS, systemic bone morphology, incorporated immunometabolic FA-species, serological markers of bone metabolism, and stress response were determined in addition to bone cell inflammation and interaction in vitro. In contrast to OA-ED, PA-ED reduced systemic bone microarchitecture paralleled by increased lipotoxic PA-containing metabolite accumulation in bone. Substitution with OA reversed the bone-destructive impact of PA, which was accompanied by reduced diacylglycerols (DAG) and saturated ceramide levels. Further, PA-associated reduction in mineralization activity and concomitant pro-inflammatory activation of primary osteoblasts were diminished in cultures where PA was replaced with OA, which impacted cellular interaction with osteoclasts. Additionally, PA-ED increased osteoclast numbers in femurs in response to oral P. gingivalis infection, whereas OA-ED reduced osteoclast occurrence, which was paralleled by serologically increased levels of the stress-reducing lipokine PI(18:1/18:1). OA substitution reverses the bone-destructive and pro-inflammatory effects of PA and eliminates incorporated lipotoxic PA metabolites. This supports Mediterranean-style OA-based diets as a preventive intervention to target the accumulation of PA-associated lipotoxic metabolites and thereby supports systemic bone tissue resilience after oral bacterial P. gingivalis infection.

Serum-Based Lipid Panels for Diagnosis of Idiopathic Parkinson's Disease.

Parkinson's disease (PD) is a highly prevalent neurodegenerative movement disorder with an unclear etiology and a lack of definite diagnostic tests and effective treatments. About 95% of PD cases are idiopathic, in which none of the well-known genes underlying familial parkinsonism are mutated. We used untargeted liquid chromatography-mass spectrometry (LC-MS/MS) to profile the serum lipidome of 50 patients with different stages of idiopathic PD (early, mid, or advanced) and 45 age-matched controls. When comparing the PD patients to the control subjects, 169 lipids were significantly altered in both a univariate analysis and a multivariate partial least-squares discriminant analysis (PLS-DA). Compared to the controls, the patients with PD had higher levels of unsaturated triacylglycerides (e.g., TG O-56:9 and TG 52:3), saturated lysophosphatidylcholines (LPC 17:0, 16:0, and 15:0), and hydroxyeicosatetraenoic acid (12-HETE), while lower levels of phosphatidylserines (e.g., PS 40:4 and PS 16:0_22:4), sphingomyelins (SM 42:1), and ceramides (e.g., Cer 40:0 and 42:0) were found between the PD patients and the controls. A panel of 10 significantly altered lipids (PS 40:0, Cer 40:0, Cer 42:0, LPC 17:0, LPC 15:0, PC 37:7, PE O-40:8, PC O-42:4, FA 23:0, and SM 42:1) resulted in a strong receiver operating characteristic curve with an AUC = 0.974. This panel may, therefore, be useful for diagnosing PD. In addition, lipid panels may prove useful for distinguishing among the progression stages of PD. Using one-way ANOVA, 155 lipid species were significantly altered among the PD stages. Parkinson's disease progressed from the early to advanced stages with decreasing levels of PC 31:1, PC 38:4, and LPE 22:5. Conversely, LPC-O 20:0, PC O-42:3, FA 19:0, and FA 22:2 showed an increase in their levels with disease progression. Overall, this study shows an intriguing number of robust changes in specific serum lipids that may become useful for diagnosing PD and its progression, once panels have been validated in larger clinical trials and prospective studies.

A defect in mitochondrial fatty acid synthesis impairs iron metabolism and causes elevated ceramide levels.

In most eukaryotic cells, fatty acid synthesis (FAS) occurs in the cytoplasm and in mitochondria. However, the relative contribution of mitochondrial FAS (mtFAS) to the cellular lipidome is not well defined. Here we show that loss of function of Drosophila mitochondrial enoyl coenzyme A reductase (Mecr), which is the enzyme required for the last step of mtFAS, causes lethality, while neuronal loss of Mecr leads to progressive neurodegeneration. We observe a defect in Fe-S cluster biogenesis and increased iron levels in flies lacking mecr, leading to elevated ceramide levels. Reducing the levels of either iron or ceramide suppresses the neurodegenerative phenotypes, indicating an interplay between ceramide and iron metabolism. Mutations in human MECR cause pediatric-onset neurodegeneration, and we show that human-derived fibroblasts display similar elevated ceramide levels and impaired iron homeostasis. In summary, this study identifies a role of mecr/MECR in ceramide and iron metabolism, providing a mechanistic link between mtFAS and neurodegeneration.

Bifidobacterium longum 68S mediated gut-skin axis homeostasis improved skin barrier damage in aging mice

BackgroundBifidobacterium as probiotics, play important roles in skin status, while the potential mechanisms interaction remains unknown. The study further explored the potential mechanism of B. longum 68S in ameliorating skin barrier damage from the perspective of the gut-skin axis in aging mice. MethodsB. longum 68S supplied natural aging mouse model and fecal microbiota transplantation (FMT) experiment proves the key role of intestinal microbiota in B. longum 68S up-regulating the production of ceramide synthesis key enzyme (SPT1) and ceramide level and improving skin barrier damage. Moreover, B. longum 68S supplied SPT1 gene deletion mouse model to investigate the mechanism of B. longum 68S on improving skin barrier damage. ResultsTranscriptome analysis and 16S rRNA high-throughput pyrosequencing demonstrated that aging mice exhibited skin barrier dysfunction and intestinal dysbiosis. Meanwhile, aging mice exhibited an up-regulation in the trans epidermal water loss (TEWL) and a down-regulation in the level of SPT1, ceramide and skin barrier-related proteins (Loricrin, Keratin 10 and Desmoglein 1). Similarity, the FMT from aging mice to normal mice and SPT1 gene deletion mice could rebuild skin barrier damage and B. longum 68S supplementation exerted a positive effect on it. Further, B. longum 68S-mediated SPT1-derived ceramide production prevented impaired ceramide synthesis-induced endoplasmic reticulum stress and apoptotic response, ultimately improving skin barrier damage in vitro. ConclusionEmerging anti-aging therapies are necessary given the poor safety profiles of current pharmaceutical drugs. B. longum 68S may be better alternatives, considering the association between the gut microbiota and healthy aging. The findings suggested that B. longum 68S-mediated gut-skin axis homeostasis, thereby exhibiting an anti-aging effect and facilitate a better understanding of the mechanisms governing the various beneficial effects of B. longum 68S.

Ceramide synthesis regulates biogenesis and packaging of exosomal MALAT1 from adipose derived stem cells, increases dermal fibroblast migration and mitochondrial function

BackgroundThe function of exosomes, small extracellular vesicles (sEV) secreted from human adipose-derived stem cells (ADSC), is becoming increasingly recognized as a means of transferring the regenerative power of stem cells to injured cells in wound healing. Exosomes are rich in ceramides and long noncoding RNA (lncRNA) like metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). We identified putative ceramide responsive cis-elements (CRCE) in MALAT1. We hypothesized that CRCE respond to cellular ceramide levels to regulate sEV MALAT1 packaging. MALAT1 levels by many cells exceed those of protein coding genes and it’s expression is equally high in exosomes. Ceramide also regulates exosome synthesis, however, the contents of exosome cargo via sphingomyelinase and ceramide synthase pathways has not been demonstrated.MethodsADSC were treated with an inhibitor of sphingomyelinase, GW4869, and stimulators of ceramide synthesis, C2- and C6-short chain ceramides, prior to collection of conditioned media (CM). sEV were isolated from CM, and then used to treat human dermal fibroblast (HDF) cultures in cell migration scratch assays, and mitochondrial stress tests to evaluate oxygen consumption rates (OCR).ResultsInhibition of sphingomyelinase by treatment of ADSC with GW4869 lowered levels of MALAT1 in small EVs. Stimulation of ceramide synthesis using C2- and C6- ceramides increased cellular, EVs levels of MALAT1. The functional role of sEV MALAT1 was evaluated in HDF by applying EVs to HDF. Control sEV increased migration of HDF, and significantly increased ATP production, basal and maximal respiration OCR. sEV from GW4869-treated ADSC inhibited cell migration and maximal respiration. However, sEV from C2- and C6-treated cells, respectively, increased both functions but not significantly above control EV except for maximal respiration. sEV were exosomes except when ADSC were treated with GW4869 and C6-ceramide, then they were larger and considered microvesicles.ConclusionsCeramide synthesis regulates MALAT1 EV content. Sphingomyelinase inhibition blocked MALAT1 from being secreted from ADSC EVs. Our report is consistent with those of MALAT1 increasing cell migration and mitochondrial MALAT1 altering maximal respiration in cells. Since MALAT1 is important for exosome function, it stands that increased exosomal MALAT1 should be beneficial for wound healing as shown with these assays.8tyb17USzV7szeprSwPAz5Video

Targeting sphingolipid metabolism with the sphingosine kinase inhibitor SKI-II overcomes hypoxia-induced chemotherapy resistance in glioblastoma cells: effects on cell death, self-renewal, and invasion

BackgroundGlioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs.MethodsDrug synergism was analyzed with the Chou-Talalay Combination Index method. Dose–effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model.ResultsTemozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids.ConclusionsThis in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.

Metabolomic profile of patients on levothyroxine treatment for hypothyroidism.

Hypothyroidism is clinically characterized by a decrease in levels of the circulating thyroid hormones namely thyroxine and triiodothyronine. The main treatment for hypothyroidism is thyroid hormone replacement using levothyroxine to normalize serum thyroid hormone levels. In this study, we explored the metabolic changes in the plasma of patients with hypothyroidism after reaching a euthyroid state with levothyroxine treatment. Plasma samples from 18 patients diagnosed as overt hypothyroidism were collected before and after levothyroxine treatment upon reaching a euthyroid state and were analyzed by high-resolution mass spectrometry-based metabolomics. Multivariate and univariate analyses evaluated data to highlight potential metabolic biomarkers. Liquid chromatography-mass spectrometry-based metabolomics revealed a significant decrease in the levels of ceramide, phosphatidylcholine, triglycerides, acylcarnitine, and peptides after levothyroxine treatment; this could indicate a change in the fatty acid transportation system and an enhanced β-oxidation, compared with a hypothyroid state. At the same time, the decrease in the peptides suggested a shift in protein synthesis. In addition, there was a considerable rise in glycocholic acid following therapy, suggesting the involvement of thyroid hormones in stimulating bile acid production and secretion. A metabolomic analysis of patients with hypothyroidism revealed significant changes in several metabolites and lipids after treatment. This study showed the value of the metabolomics technique in providing a complementary understanding of the pathophysiology of hypothyroidism and as a crucial tool for examining the molecular impact of levothyroxine treatment on hypothyroidism. It was an important tool for investigating the therapeutic effects of levothyroxine on hypothyroidism at the molecular level.

Effect of activated glycyrrhizic acid on epithelial cell lipidome in HPV-associated cervical lesions: A prospective cohort study

Aim. To determine the change of lipid levels in the cervical epithelial cells during the treatment of chronic cervicitis and low-grade squamous intraepithelial lesions (LSIL) with 0.1% activated glycyrrhizic acid.
 Materials and methods. The prospective cohort study included 40 patients with chronic cervicitis and 40 patients with LSIL. Cytological evaluation of cervical smears was performed according to the Bethesda system (2014). All patients received Epigen Intim Spray for 18 months. Amplification of type-specific DNA fragments of human papillomavirus (HPV) and human DNA (sampling control SC) was done using a kit of reagents to detect, type, and quantitatively determine 21 HPV types by Quantum-21 HPV PCR. Lipid extract of a cervical epithelial scraping taken before and after therapy was analyzed by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Lipids were identified using the Lipid Match R-script and by characteristic MS/MS. Correlation analysis of the lipidome of cervicovaginal fluid and SC was performed using the Spearman test, and the Mann-Whitney test was used to compare lipid levels at the two time points.
 Results. During the treatment of chronic cervicitis, the levels of CL lipids 16:0_16:1_18:1_18:1, HexCer-NDS d20:0/26:0, PC 16: 0_18: 1 and 16: 0_20:4 were decreasing; during the treatment of LSIL, the levels of ceramides and glucosylceramides were decreasing. Chronic cervicitis is characterized by a positive correlation of SC smear with cardiolipins, glucosylceramides, and epithelial lipids with an ether bond; LSIL showed a negative correlation with phosphatidylcholines and phosphatidylethanolamines with an ether bond.
 Conclusion. As a result of treatment with activated glycyrrhizic acid, the lipid spectrum of epithelial cells significantly changed, depending on the type of lesion. It is also necessary to consider SC scrapings in further studies of epithelial cell lipidome and the type of HPV-associated lesions that affects the adjustments.

The Interplay between Bioactive Sphingolipids in the Psoriatic Skin and the Severity of the Disease.

Psoriasis is a complex chronic immunologically mediated disease that may involve skin, nails, and joints. It is characterized by hyperproliferation, deregulated differentiation, and impaired apoptosis of keratinocytes. Sphingolipids, namely ceramide, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate, are signal molecules that may regulate cell growth, immune reactions, and apoptosis. Fifteen patients with psoriasis and seventeen healthy persons were enrolled in the study. Skin samples were taken from psoriatic lesions and non-lesional areas. Tissue concentration of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate was measured by liquid chromatography. We assessed that all levels of ceramides, sphingosine-1-phosphate, sphingosine, sphingomyelin, and sphinganine-1-phosphate were higher in lesioned psoriatic skin than in non-affected skin. The profile of bioactive lipids in the lesional skin of patients with psoriasis differed significantly from non-involved psoriatic skin and skin in healthy subjects.

Multi-Omic Integration of Blood-Based Tumor-Associated Genomic and Lipidomic Profiles Using Machine Learning Models in Metastatic Prostate Cancer.

To determine prognostic and predictive clinical outcomes in metastatic hormone-sensitive prostate cancer (mHSPC) and metastatic castrate-resistant prostate cancer (mCRPC) on the basis of a combination of plasma-derived genomic alterations and lipid features in a longitudinal cohort of patients with advanced prostate cancer. A multifeature classifier was constructed to predict clinical outcomes using plasma-based genomic alterations detected in 120 genes and 772 lipidomic species as informative features in a cohort of 71 patients with mHSPC and 144 patients with mCRPC. Outcomes of interest were collected over 11 years of follow-up. These included in mHSPC state early failure of androgen-deprivation therapy (ADT) and exceptional responders to ADT; early death (poor prognosis) and long-term survivors in mCRPC state. The approach was to build binary classification models that identified discriminative candidates with optimal weights to predict outcomes. To achieve this, we built multi-omic feature-based classifiers using traditional machine learning (ML) methods, including logistic regression with sparse regularization, multi-kernel Gaussian process regression, and support vector machines. The levels of specific ceramides (d18:1/14:0 and d18:1/17:0), and the presence of CHEK2 mutations, AR amplification, and RB1 deletion were identified as the most crucial factors associated with clinical outcomes. Using ML models, the optimal multi-omics feature combination determined resulted in AUC scores of 0.751 for predicting mHSPC survival and 0.638 for predicting ADT failure; and in mCRPC state, 0.687 for prognostication and 0.727 for exceptional survival. The models were observed to be superior than using a limited candidate number of features for developing multi-omic prognostic and predictive signatures. Using a ML approach that incorporates multiple omic features improves the prediction accuracy for metastatic prostate cancer outcomes significantly. Validation of these models will be needed in independent data sets in future.

A localized hydrogel-mediated chemotherapy causes immunogenic cell death via activation of ceramide-mediated unfolded protein response.

Treatment of triple-negative breast cancer (TNBC) is challenging because of its "COLD" tumor immunosuppressive microenvironment (TIME). Here, we present a hydrogel-mediated localized delivery of a combination of docetaxel (DTX) and carboplatin (CPT) (called DTX-CPT-Gel therapy) that ensured enhanced anticancer effect and tumor regression on multiple murine syngeneic and xenograft tumor models. DTX-CPT-Gel therapy modulated the TIME by an increase of antitumorigenic M1 macrophages, attenuation of myeloid-derived suppressor cells, and increase of granzyme B+CD8+ T cells. DTX-CPT-Gel therapy elevated ceramide levels in tumor tissues that activated the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK)-mediated unfolded protein response (UPR). This UPR-mediated activation of apoptotic cell death led to release of damage-associated molecular patterns, thereby activating the immunogenic cell death that could even clear the metastatic tumors. This study provides a promising hydrogel-mediated platform for DTX-CPT therapy that induces tumor regression and effective immune modulation and, therefore, can be explored further for treatment of TNBC.

Chronic nSMase inhibition suppresses neuronal exosome spreading and sex-specifically attenuates amyloid pathology in APP knock-in Alzheimer's disease mice

Female biased pathology and cognitive decline in Alzheimer's disease (AD) have been consistently observed with unclear underlying mechanisms. Although brain sphingolipid ceramide is elevated in AD patients, whether and how ceramide may contribute to sex-specific differences in amyloid pathology is unknown. Here we investigated the sex-specific impact of chronic pharmacological inhibition of neutral sphingomyelinase (nSMase), a key enzyme responsible for ceramide metabolism, on in vivo neuron-derived exosome dynamics, Aβ plaque load, and cognitive function in the APPNL-F/NL-F knock-in (APP NL-F) AD mouse model. Our results found sex-specific increase of cortical C20:0 ceramide and brain exosome levels only in APP NL-F but not in age-matched WT mice. Although nSMase inhibition similarly blocks exosome spreading in male and female mice, significantly reduced amyloid pathology was mostly observed in cortex and hippocampus of female APP NL-F mice with only modest effect found on male APP NL-F mice. Consistently, T maze test to examine spatial working memory revealed a female-specific reduction in spontaneous alternation rate in APP NL-F mice, which was fully reversed with chronic nSMase inhibition. Together, our results suggest that disease induced changes in ceramide and exosome pathways contribute to the progression of female-specific amyloid pathology in APP NL-F AD models.

An exploratory study of plasma ceramides in comorbidities in Down syndrome.

Plasma ceramide levels (henceforth, "ceramides") are biomarkers of some diseases that are comorbidities of Down syndrome (DS). We sought to determine if comorbidities in DS were associated with ceramides, studying a convenience cohort of 35 study participants, all ≥12 months old. To identify comorbidities, we reviewed the problem lists in electronic health records that were concurrent with sample collection. We placed clinically related comorbidities into one of five categories of comorbidities, henceforth, categories: obesity/overweight; autoimmune disease; congenital heart disease; bacterial infection; and central nervous system (CNS) condition. We measured the eight ceramides most frequently associated with disease using liquid chromatography-tandem mass spectrometry. We calculated a ceramide composite outcome score (CCOS) for each participant by normalizing each ceramide level to the mean for that level in the study population and then summing the normalized levels, to be proxy variable for all eight ceramides in aggregate. We used multivariable linear regression models adjusted for age and sex to test associations of categories with ceramides and with CCOSs. Post hoc, we realized that co-occurring comorbidities might interfere with establishing associations between predictor categories and ceramides and that stratified analyses might eliminate their influence on associations. We posited that CCOSs could be used to screen for associations of categories with multiple ceramides, since most diseases have been associated with more than one ceramide. We chose to omit in the stratified analyses the two categories that were the most different from one another in their associations with their CCOSs, having the most divergent regression coefficients (the highest positive and lowest negative coefficients). We first omitted one of these two divergent categories in a stratified analysis and tested in the remaining participants (those without a comorbidity in the interfering category) for associations of the other four categories with their CCOSs and then did the same for the other divergent category. In each of these two screening stratified analyses, we found one category was significantly associated with its CCOS. In the two identified categories, we then tested for associations with each of the eight ceramides, using the appropriate stratified analysis. Next, we sought to determine if the associations of the two categories with ceramides we found by omitting participants in the interfering categories held in our small sample for participants in the omitted categories as well. For each of the two categories, we therefore omitted participants without the interfering category and determined associations between the predictor category and individual ceramides in the remaining participants (those with a comorbidity in the interfering category). In the a priori analyses, autoimmune disease was inversely associated with C16 and CNS condition was inversely associated with C23. Obesity/overweight and CNS condition were the two categories with the most divergent regression coefficients (0.037 vs. -0.048). In post hoc stratified analyses, after omitting participants with obesity/overweight, thereby leaving participants without obesity/overweight, bacterial infection was associated with its CCOS and then with C14, C20, and C22. However, in the companion stratified analyses, omitting participants without obesity/overweight, thereby leaving participants with obesity/overweight, bacterial infection was not associated with any of the eight ceramides. Similarly, in post hoc stratified analyses after omitting participants with a CNS condition, thereby leaving participants without a CNS condition, obesity/overweight was associated with its CCOS and then with C14, C23, and C24. In the companion analyses, omitting participants without a CNS condition, thereby leaving participants with a CNS condition, obesity/overweight was inversely associated with C24.1. In conclusion, CNS and autoimmune disease were inversely associated with one ceramide each in a priori analyses. In post hoc analyses, we serendipitously omitted categories that interfered with associations of other categories with ceramides in stratified analyses. We found that bacterial infection was associated with three ceramides in participants without obesity/overweight and that obesity/overweight was associated with three ceramides in participants without a CNS condition. We therefore identified obesity/overweight and CNS conditions as potential confounders or effect modifiers for these associations. This is the first report of ceramides in DS and in human bacterial infection. Further study of ceramides in comorbidities of DS is justified.

1638-P: Oxidized Lipids Cause Ceramide Accumulation and Insulin Resistance in Skeletal Muscle

Accumulation of muscle lipids promotes insulin resistance and type 2 diabetes (T2D). Ceramides are important muscle lipids that decrease insulin sensitivity and it was previously shown that accumulation of ceramides in the sarcolemma negatively impacts insulin sensitivity in humans. The mechanisms explaining accumulation of lipids in specific compartments in skeletal muscle are unknown, but one possibility is through circulating oxidized lipids. POVPC, a major circulating oxidized phospholipid, was exposed to rat myotubes (1-50 μM) for 4h and the total cellular ceramide levels in myotubes measured by LC-MS/MS. Total ceramide content increased dose dependently at POVPC doses in the physiological range (5 μM, p <0.05). To determine the mechanism of POVPC induced ceramide accumulation, ceramide was measured in myotubes after treatment with POVPC (25 μM) in the presence of inhibitors of neutral and acid sphingomyelinase (GW4869 and ARC39), ceramide synthase (fumonisin B1) and serine palmitoyltransferase (myriocin). The individually administered inhibitors in the presence of POVPC each showed a decrease (41-55%) in ceramide accumulation. When all of inhibitors were administered together along with POVPC, an almost complete inhibition of ceramide accumulation (95%) was observed indicating that the sphingomyelinase, de novo, and salvage pathways of ceramide synthesis are activated by POVPC. The role of oxidized lipids on insulin sensitivity was examined after treatment of rat myotubes with 1-25 μM POVPC. Increasing doses of POVPC at doses as low as 1 μM significantly decreased insulin sensitivity in vitro (p <0.05). These results reveal circulating oxidized lipids promote ceramide accumulation and insulin resistance in muscle cells and may help explain variability in total and localized muscle ceramide levels between individuals. These data also suggest that therapies designed to lower circulating oxidized lipids could be beneficial for the treatment and prevention of T2D. Disclosure K.Zemski berry: None. B.C.Bergman: Research Support; Eli Lilly and Company. Funding National Institutes of Health (P30DK048520); University of Colorado (25N0350)

Solid tumor treatment via augmentation of bioactive C6 ceramide levels with thermally ablative focused ultrasound.

Sparse scan partial thermal ablation (TA) with focused ultrasound (FUS) may be deployed to treat solid tumors and increase delivery of systemically administered therapeutics. Furthermore, C6-ceramide-loaded nanoliposomes (CNLs), which rely upon the enhanced-permeation and retention (EPR) effect for delivery, have shown promise for treating solid tumors and are being tested in clinical trials. Here, our objective was to determine whether CNLs synergize with TA in the control of 4T1 breast tumors. CNL monotherapy of 4T1 tumors yielded significant intratumoral bioactive C6 accumulation by the EPR effect, but tumor growth was not controlled. TA increased bioactive C6 accumulation by ~ 12.5-fold over the EPR effect. In addition, TA + CNL caused shifts in long-chain to very-long-chain ceramide ratios (i.e., C16/24 and C18/C24) that could potentially contribute to tumor control. Nonetheless, these changes in intratumoral ceramide levels were still insufficient to confer tumor growth control beyond that achieved when combining with TA with control "ghost" nanoliposomes (GNL). While this lack of synergy could be due to increased "pro-tumor" sphingosine-1-phosphate (S1P) levels, this is unlikely because S1P levels exhibited only a moderate and statistically insignificant increase with TA + CNL. In vitro studies showed that 4T1 cells are highly resistant to C6, offering the most likely explanation for the inability of TA to synergize with CNL. Thus, while our results show that sparse scan TA is a powerful approach for markedly enhancing CNL delivery and generating "anti-tumor" shifts in long-chain to very-long-chain ceramide ratios, resistance of the tumor to C6 can still be a rate-limiting factor for some solid tumor types.

Metabolomic signature and molecular profile of normal and degenerated human intervertebral disc cells

BACKGROUND CONTEXTIntervertebral disc degeneration (IVDD) is an incurable, specific treatment-orphan disease with an increasing burden worldwide. Although great efforts have been made to develop new regenerative therapies, their clinical success is limited. PURPOSECharacterize the metabolomic and gene expression changes underpinning human disc degeneration. This study also aimed to disclose new molecular targets for developing and optimizing novel biological approaches for IVDD. STUDY DESIGNIntervertebral disc cells were obtained from IVDD patients undergoing circumferential arthrodesis surgery or from healthy subjects. Mimicking the harmful microenvironment of degenerated discs, cells isolated from the nucleus pulposus (NP) and annulus fibrosus (AF) were exposed to the proinflammatory cytokine IL-1β and the adipokine leptin. The metabolomic signature and molecular profile of human disc cells were unraveled for the first time. METHODSThe metabolomic and lipidomic profiles of IVDD and healthy disc cells were analyzed by high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Gene expression was investigated by SYBR green-based quantitative real-time RT-PCR. Altered metabolites and gene expression were documented. RESULTSLipidomic analysis revealed decreased levels of triacylglycerols (TG), diacylglycerol (DG), fatty acids (FA), phosphatidylcholine (PC), lysophosphatidylinositols (LPI) and sphingomyelin (SM), and increased levels of bile acids (BA) and ceramides, likely promoting disc cell metabolism changing from glycolysis to fatty acid oxidation and following cell death. The gene expression profile of disc cells suggests LCN2 and LEAP2/GHRL as promising molecular therapeutic targets for disc degeneration and demonstrates the expression of genes related to inflammation (NOS2, COX2, IL-6, IL-8, IL-1β, and TNF-α) or encoding adipokines (PGRN, NAMPT, NUCB2, SERPINE2, and RARRES2), matrix metalloproteinases (MMP9 and MMP13), and vascular adhesion molecules (VCAM1). CONCLUSIONSAltogether, the presented results disclose the NP and AF cell biology changes from healthy to degenerated discs, allowing the identification of promising molecular therapeutic targets for intervertebral disc degeneration. CLINICAL SIGNIFICANCEOur results are relevant to improving current biological-based strategies aiming to repair IVD by restoring cellular lipid metabolites as well as adipokines homeostasis. Ultimately, our results will be valuable for successful, long-lasting relief of painful IVDD.

Identification of predictive biomarkers for diagnosis and radiation sensitivity of uterine cervical cancer using wide-targeted metabolomics.

Uterine cervical cancer (UCC) is the fourth most common cancer in women, responsible for more than 300 000 deaths worldwide. Its early detection, by cervical cytology, and prevention, by vaccinating against human papilloma virus, greatly contribute to reducing cervical cancer mortality in women. However, penetration of the effective prevention of UCC in Japan remains low. Plasma metabolome analysis is widely used for biomarker discovery and the identification of cancer-specific metabolic pathways. Here, we aimed to identify predictive biomarkers for the diagnosis and radiation sensitivity of UCC using wide-targeted plasma metabolomics. We analyzed 628 metabolites in plasma samples obtained from 45 patients with UCC using ultra-high-performance liquid chromatography with tandem mass spectrometry. The levels of 47 metabolites were significantly increased and those of 75 metabolites were significantly decreased in patients with UCC relative to healthy controls. Increased levels of arginine and ceramides, and decreased levels of tryptophan, ornithine, glycosylceramides, lysophosphatidylcholine, and phosphatidylcholine were characteristic of patients with UCC. Comparison of metabolite profiles in groups susceptible and non-susceptible to radiation therapy, a treatment for UCC, revealed marked variations in polyunsaturated fatty acid, nucleic acid, and arginine metabolism in the group not susceptible to treatment. Our findings suggest that the metabolite profile of patients with UCC may be an important indicator for distinguishing these patients from healthy cohorts, and may also be useful for predicting sensitivity to radiotherapy.

Impact of HepG2 Cells Glutathione Depletion on Neutral Sphingomyelinases mRNA Levels and Activity

Liver cancer is a prevalent form of cancer worldwide. While research has shown that increasing sphingomyelin (SM) hydrolysis by activating the cell surface membrane-associated neutral sphingomyelinase 2 (nSMase2) can control cell proliferation and apoptosis, the role of total glutathione depletion in inducing tumor cell apoptosis via nSMase2 activation is still under investigation. Conversely, glutathione-mediated inhibition of reactive oxygen species (ROS) accumulation is necessary for the enzymatic activity of nSMase1 and nSMase3, increased ceramide levels, and cell apoptosis. This study evaluated the effects of depleting total glutathione in HepG2 cells using buthionine sulfoximine (BSO). The study assessed nSMases RNA levels and activities, intracellular ceramide levels, and cell proliferation using RT-qPCR, Amplex red neutral sphingomyelinase fluorescence assay, and colorimetric assays, respectively. The results indicated a lack of nSMase2 mRNA expression in treated and untreated HepG2 cells. Depletion of total glutathione resulted in a significant increase in mRNA levels but a dramatic reduction in the enzymatic activity of nSMase1 and nSMase3, a rise in ROS levels, a decrease in intracellular levels of ceramide, and an increase in cell proliferation. These findings suggest that total glutathione depletion may exacerbate liver cancer (HCC) and not support using total glutathione-depleting agents in HCC management. It is important to note that these results are limited to HepG2 cells, and further studies are necessary to determine if these effects will also occur in other cell lines. Additional research is necessary to explore the role of total glutathione depletion in inducing tumor cell apoptosis.

Integrated microbiomic and metabolomic analyses reveal the mechanisms by which bee pollen and royal jelly lipid extracts ameliorate colitis in mice

Bee pollen (BP) and royal jelly (RJ) have shown therapeutic effects against colitis, but the functional components contained therein remain elusive. Here, we used an integrated microbiomic-metabolomic strategy to clarify the mechanism by which bee pollen lipid extracts (BPL) and royal jelly lipid extracts (RJL) ameliorated dextran sulfate sodium (DSS)-induced colitis in mice. Lipidomic results showed that levels of ceramide (Cer), lysophosphatidylcholine (LPC), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were significantly higher in BPL than in RJL. The anti-inflammatory efficacy of BPL surpassed that of RJL, although both BPL and RJL could attenuate DSS-induced colitis through several mechanisms: reducing the disease activity index (DAI); decreasing histopathological damage; inhibiting the expression of genes encoding proinflammatory cytokines; improving intestinal microbial community structure, and modulating host metabolism. These findings demonstrated that BPL and RJL have great potential as functional ingredients for the production of dietary supplements to prevent early colitis.