Justin Snider

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• Buss, L. G., De Oliveira Pessoa, D., Snider, J. M., Padi, M., Martinez, J. A., & Limesand, K. H. (2023). Metabolomics analysis of pathways underlying radiation-induced salivary gland dysfunction stages. PloS one, 18(11), e0294355.
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  Salivary gland hypofunction is an adverse side effect associated with radiotherapy for head and neck cancer patients. This study delineated metabolic changes at acute, intermediate, and chronic radiation damage response stages in mouse salivary glands following a single 5 Gy dose. Ultra-high performance liquid chromatography-mass spectrometry was performed on parotid salivary gland tissue collected at 3, 14, and 30 days following radiation (IR). Pathway enrichment analysis, network analysis based on metabolite structural similarity, and network analysis based on metabolite abundance correlations were used to incorporate both metabolite levels and structural annotation. The greatest number of enriched pathways are observed at 3 days and the lowest at 30 days following radiation. Amino acid metabolism pathways, glutathione metabolism, and central carbon metabolism in cancer are enriched at all radiation time points across different analytical methods. This study suggests that glutathione and central carbon metabolism in cancer may be important pathways in the unresolved effect of radiation treatment.
• Chauhan, S. S., Casillas, A. L., Vizzerra, A. D., Liou, H., Clements, A. N., Flores, C. E., Prevost, C. T., Kashatus, D. F., Snider, A. J., Snider, J. M., & Warfel, N. A. (2023). PIM1 drives lipid droplet accumulation to promote proliferation and survival in prostate cancer. Oncogene.
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  Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer. Mechanistically, PIM1 inactivates glycogen synthase kinase 3 beta (GSK3β) via serine 9 phosphorylation. GSK3β inhibition stabilizes PPARα and enhances the transcription of genes linked to peroxisomal biogenesis (PEX3 and PEX5) and LD growth (Tip47). The effects of PIM1 on LD accumulation are abrogated with GW6471, a specific inhibitor for PPARα. Notably, LD accumulation downstream of PIM1 provides a significant survival advantage for prostate cancer cells during nutrient stress, such as glucose depletion. Inhibiting PIM reduces LD accumulation in vivo alongside slow tumor growth and proliferation. Furthermore, TKO mice, lacking PIM isoforms, exhibit suppression in circulating triglycerides. Overall, our findings establish PIM1 as an important regulator of LD accumulation through GSK3β-PPARα signaling axis to promote cell proliferation and survival during nutrient stress.
• Espinoza, K. S., Hermanson, K. N., Beard, C. A., Schwartz, N. U., Snider, J. M., Low, B. E., Wiles, M. V., Hannun, Y. A., Obeid, L. M., & Snider, A. J. (2023). A novel HSPB1 mouse model of Charcot-Marie-Tooth Disease. Prostaglandins & other lipid mediators, 169, 106769.
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  Charcot-Marie-Tooth Disease (CMT) is a commonly inherited peripheral polyneuropathy. Clinical manifestations for this disease include symmetrical distal polyneuropathy, altered deep tendon reflexes, distal sensory loss, foot deformities, and gait abnormalities. Genetic mutations in heat shock proteins have been linked to CMT2. Specifically, mutations in the heat shock protein B1 (HSPB1) gene encoding for heat shock protein 27 (Hsp27) have been linked to CMT2F and distal hereditary motor and sensory neuropathy type 2B (dHMSN2B) subtype. The goal of the study was to examine the role of an endogenous mutation in HSPB1 in vivo and to define the effects of this mutation on motor function and pathology in a novel animal model. As sphingolipids have been implicated in hereditary and sensory neuropathies, we examined sphingolipid metabolism in central and peripheral nervous tissues in 3-month-old Hsp mice. Though sphingolipid levels were not altered in sciatic nerves from Hsp mice, ceramides and deoxyceramides, as well as sphingomyelins (SMs) were elevated in brain tissues from Hsp mice. Histology was utilized to further characterize Hsp mice. Hsp mice exhibited no alterations to the expression and phosphorylation of neurofilaments, or in the expression of acetylated α-tubulin in the brain or sciatic nerve. Interestingly, Hsp mice demonstrated cerebellar demyelination. Locomotor function, grip strength and gait were examined to define the role of Hsp in the clinical phenotypes associated with CMT2F. Gait analysis revealed no differences between Hsp and Hsp mice. However, both coordination and grip strength were decreased in 3-month-old Hsp mice. Together these data suggest that the endogenous S139F mutation in HSPB1 may serve as a mouse model for hereditary and sensory neuropathies such as CMT2F.
• Furlong, M. A., Liu, T., Snider, J. M., Tfaily, M. M., Itson, C., Beitel, S., Parsawar, K., Keck, K., Galligan, J., Walker, D. I., Gulotta, J. J., & Burgess, J. L. (2023). Evaluating changes in firefighter urinary metabolomes after structural fires: an untargeted, high resolution approach. Scientific reports, 13(1), 20872.
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  Firefighters have elevated rates of urinary tract cancers and other adverse health outcomes, which may be attributable to environmental occupational exposures. Untargeted metabolomics was applied to characterize this suite of environmental exposures and biological changes in response to occupational firefighting. 200 urine samples from 100 firefighters collected at baseline and two to four hours post-fire were analyzed using untargeted liquid-chromatography and high-resolution mass spectrometry. Changes in metabolite abundance after a fire were estimated with fixed effects linear regression, with false discovery rate (FDR) adjustment. Partial least squares discriminant analysis (PLS-DA) was also used, and variable important projection (VIP) scores were extracted. Systemic changes were evaluated using pathway enrichment for highly discriminating metabolites. Metabolome-wide-association-study (MWAS) identified 268 metabolites associated with firefighting activity at FDR q 
• Richardson, W. J., Humphrey, S. B., Sears, S. M., Hoffman, N. A., Orwick, A. J., Doll, M. A., Doll, C. L., Xia, C., Hernandez-Corbacho, M., Snider, J. M., Obeid, L. M., Hannun, Y. A., Snider, A. J., & Siskind, L. J. (2023).

  Expression of ceramide synthases in mice and their roles in regulating acyl-chain sphingolipids: A framework for baseline levels and future implications in aging and disease

  . Molecular pharmacology.
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  Sphingolipids are an important class of lipids present in all eukaryotic cells that regulate critical cellular processes. Disturbances in sphingolipid homeostasis have been linked to several diseases in humans. Ceramides are central in sphingolipid metabolism and are largely synthesized by six ceramide synthase isoforms (CerS1-6), each with a preference for different fatty acyl chain lengths. While the tissue distribution of CerS mRNA expression in humans and the roles of CerS isoforms in synthesizing ceramides with different acyl chain lengths are known, it is unknown how CerS expression dictates ceramides and downstream metabolites within tissues. In this study, we analyzed sphingolipid levels and CerS mRNA expression in 3-month-old C57BL/6J mouse brain, heart, kidney, liver, lung, and skeletal muscle. The results showed that CerS expression and sphingolipid species abundance varied by tissue and that CerS expression was a predictor of ceramide species within tissues. Interestingly, though CerS expression was not predictive of complex sphingolipid species within all tissues, composite scores for CerSs contributions to total sphingolipids measured in each tissue correlated to CerS expression. Lastly, we determined that the most abundant ceramide species in mouse tissues aligned with CerS mRNA expression in corresponding human tissues (based on chain length preference), suggesting mice are relevant preclinical models for ceramide and sphingolipid research. The current study demonstrates that CerS expression in specific tissues correlates not only with ceramide species but contributes to the generation of complex sphingolipids as well. As many of the CerSs and/ or specific ceramide species have been implicated in disease, these studies suggest the potential for CerSs as therapeutic targets and the use of sphingolipid species as diagnostics in specific tissues.
• Linzer, R. W., Guida, D. L., Aminov, J., Snider, J. M., Khalife, G., Buyukbayraktar, A. B., Alhaddad, C., Resnick, A. E., Wang, P., Pan, C. H., Allopenna, J. J., & Clarke, C. J. (2022). Dihydroceramide desaturase 1 (DES1) promotes anchorage-independent survival downstream of HER2-driven glucose uptake and metabolism. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 36(10), e22558.
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  Oncogenic reprogramming of cellular metabolism is a hallmark of many cancers, but our mechanistic understanding of how such dysregulation is linked to tumor behavior remains poor. In this study, we have identified dihydroceramide desaturase (DES1)-which catalyzes the last step in de novo sphingolipid synthesis-as necessary for the acquisition of anchorage-independent survival (AIS), a key cancer enabling biology, and establish DES1 as a downstream effector of HER2-driven glucose uptake and metabolism. We further show that DES1 is sufficient to drive AIS and in vitro tumorigenicity and that increased DES1 levels-found in a third of HER2+ breast cancers-are associated with worse survival outcomes. Taken together, our findings reveal a novel pro-tumor role for DES1 as a transducer of HER2-driven glucose metabolic signals and provide evidence that targeting DES1 is an effective approach for overcoming AIS. Results further suggest that DES1 may have utility as a biomarker of aggressive and metastasis-prone HER2+ breast cancer.
• Lu, E., Hara, A., Sun, S., Hallmark, B., Snider, J. M., Seeds, M. C., Watkins, J. C., McCall, C. E., Zhang, H. H., Yao, G., & Chilton, F. H. (2022). Temporal Associations of Plasma Levels of the Secreted Phospholipase A Family and Mortality in Severe COVID-19. medRxiv : the preprint server for health sciences.
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  Previous research suggests that group IIA secreted phospholipase A (sPLA -IIA) plays a role in and predicts severe COVID-19 disease. The current study reanalyzed a longitudinal proteomic data set to determine the temporal (days 0, 3 and 7) relationship between the levels of several members of a family of sPLA isoforms and the severity of COVID-19 in 214 ICU patients. The levels of six secreted PLA isoforms, sPLA -IIA, sPLA -V, sPLA -X, sPLA -IB, sPLA -IIC, and sPLA -XVI, increased over the first 7 ICU days in those who succumbed to the disease. sPLA -IIA outperformed top ranked cytokines and chemokines as predictors of patient outcome. A decision tree corroborated these results with day 0 to day 3 kinetic changes of sPLA -IIA that separated the death and severe categories from the mild category and increases from day 3 to day 7 significantly enriched the lethal category. In contrast, there was a time-dependent decrease in sPLA -IID and sPLA -XIIB in patients with severe or lethal disease, and these two isoforms were at higher levels in mild patients. Taken together, proteomic analysis revealed temporal sPLA patterns that reflect the critical roles of sPLA isoforms in severe COVID-19 disease.
• Bouchareb, R., Guauque-Olarte, S., Snider, J., Zaminski, D., Anyanwu, A., Stelzer, P., & Lebeche, D. (2021). Proteomic Architecture of Valvular Extracellular Matrix: FNDC1 and MXRA5 Are New Biomarkers of Aortic Stenosis. JACC. Basic to translational science, 6(1), 25-39.
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  This study analyzed the expression of extracellular matrix (ECM) proteins during aortic valve calcification with mass spectrometry, and further validated in an independent human cohort using RNAseq data. The study reveals that valve calcification is associated with significant disruption in ECM and metabolic pathways, and highlights a strong connection between metabolic markers and ECM remodeling. It also identifies FNDC1 and MXRA5 as novel ECM biomarkers in calcified valves, electing them as potential targets in the development and progression of aortic stenosis.
• Otsuka, Y., Airola, M. V., Choi, Y. M., Coant, N., Snider, J., Cariello, C., Saied, E. M., Arenz, C., Bannister, T., Rahaim, R., Hannun, Y. A., Shumate, J., Scampavia, L., Haley, J. D., & Spicer, T. P. (2021). Identification of Small-Molecule Inhibitors of Neutral Ceramidase (nCDase) via Target-Based High-Throughput Screening. SLAS discovery : advancing life sciences R & D, 26(1), 113-121.
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  There is interest in developing inhibitors of human neutral ceramidase (nCDase) because this enzyme plays a critical role in colon cancer. There are currently no potent or clinically effective inhibitors for nCDase reported to date, so we adapted a fluorescence-based enzyme activity method to a high-throughput screening format. We opted to use an assay whereby nCDase hydrolyzes the substrate RBM 14-16, and the addition of NaIO4 acts as an oxidant that releases umbelliferone, resulting in a fluorescent signal. As designed, test compounds that act as ceramidase inhibitors will prevent the hydrolysis of RBM 14-16, thereby decreasing fluorescence. This assay uses a 1536-well plate format with excitation in the blue spectrum of light energy, which could be a liability, so we incorporated a counterscreen that allows for rapid selection against fluorescence artifacts to minimize false-positive hits. The high-throughput screen of >650,000 small molecules found several lead series of hits. Multiple rounds of chemical optimization ensued with improved potency in terms of IC and selectivity over counterscreen assays. This study describes the first large-scale high-throughput optical screening assay for nCDase inhibitors that has resulted in leads that are now being pursued in crystal docking studies and in vitro drug metabolism and pharmacokinetics (DMPK).
• Selmin, O. I., Papoutsis, A. J., Hazan, S., Smith, C., Greenfield, N., Donovan, M. G., Wren, S. N., Doetschman, T. C., Snider, J. M., Snider, A. J., Chow, S. H., & Romagnolo, D. F. (2021). -6 High Fat Diet Induces Gut Microbiome Dysbiosis and Colonic Inflammation. International journal of molecular sciences, 22(13).
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  : Concerns are emerging that a high-fat diet rich in -6 PUFA (-6HFD) may alter gut microbiome and increase the risk of intestinal disorders. Research is needed to model the relationships between consumption of an -6HFD starting at weaning and development of gut dysbiosis and colonic inflammation in adulthood. We used a C57BL/6J mouse model to compare the effects of exposure to a typical American Western diet (WD) providing 58.4%, 27.8%, and 13.7% energy (%E) from carbohydrates, fat, and protein, respectively, with those of an isocaloric and isoproteic soybean oil-rich -6HFD providing 50%E and 35.9%E from total fat and carbohydrates, respectively on gut inflammation and microbiome profile. : At weaning, male offspring were assigned to either the WD or -6HFD through 10-16 weeks of age. The WD included fat exclusively from palm oil whereas the -6HFD contained fat exclusively from soybean oil. We recorded changes in body weight, cyclooxygenase-2 (COX-2) expression, colon histopathology, and gut microbiome profile. : Compared to the WD, the -6HFD increased plasma levels of -6 fatty acids; colonic expression of COX-2; and the number of colonic inflammatory and hyperplastic lesions. At 16 weeks of age, the -6HFD caused a marked reduction in the gut presence of , , and , and induced growth of and . At the species level, the -6HFD sustains the gut growth of proinflammatory and . : An -6HFD consumed from weaning to adulthood induces a shift in gut bacterial profile associated with colonic inflammation.
• Snider, J. M., Trayssac, M., Clarke, C. J., Schwartz, N., Snider, A. J., Obeid, L. M., Luberto, C., & Hannun, Y. A. (2021). Erratum: Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis. Journal of lipid research, 62, 100009.
• Snider, J. M., You, J. K., Wang, X., Snider, A. J., Hallmark, B., Seeds, M. C., Sergeant, S., Johnstone, L., Wang, Q., Sprissler, R., Zhang, H. H., Luberto, C., Kew, R. R., Hannun, Y. A., McCall, C. E., Yao, G., Del Poeta, M., & Chilton, F. H. (2021). Group IIA Secreted Phospholipase A Plays a Central Role in the Pathobiology of COVID-19. medRxiv : the preprint server for health sciences.
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  There is an urgent need to identify cellular and molecular mechanisms responsible for severe COVID-19 disease accompanied by multiple organ failure and high mortality rates. Here, we performed untargeted/targeted lipidomics and focused biochemistry on 127 patient plasma samples, and showed high levels of circulating, enzymatically active secreted phospholipase A Group IIA (sPLA -IIA) in severe and fatal COVID-19 disease compared with uninfected patients or mild illness. Machine learning demonstrated that sPLA -IIA effectively stratifies severe from fatal COVID-19 disease. We further introduce a PLA-BUN index that combines sPLA -IIA and blood urea nitrogen (BUN) threshold levels as a critical risk factor for mitochondrial dysfunction, sustained inflammatory injury and lethal COVID-19. With the availability of clinically tested inhibitors of sPLA -IIA, our study opens the door to a precision intervention using indices discovered here to reduce COVID-19 mortality.
• Snider, J. M., You, J. K., Wang, X., Snider, A. J., Hallmark, B., Zec, M. M., Seeds, M. C., Sergeant, S., Johnstone, L., Wang, Q., Sprissler, R., Carr, T. F., Lutrick, K., Parthasarathy, S., Bime, C., Zhang, H. H., Luberto, C., Kew, R. R., Hannun, Y. A., , Guerra, S., et al. (2021). Group IIA secreted phospholipase A2 is associated with the pathobiology leading to COVID-19 mortality. The Journal of clinical investigation, 131(19).
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  There is an urgent need to identify the cellular and molecular mechanisms responsible for severe COVID-19 that results in death. We initially performed both untargeted and targeted lipidomics as well as focused biochemical analyses of 127 plasma samples and found elevated metabolites associated with secreted phospholipase A2 (sPLA2) activity and mitochondrial dysfunction in patients with severe COVID-19. Deceased COVID-19 patients had higher levels of circulating, catalytically active sPLA2 group IIA (sPLA2-IIA), with a median value that was 9.6-fold higher than that for patients with mild disease and 5.0-fold higher than the median value for survivors of severe COVID-19. Elevated sPLA2-IIA levels paralleled several indices of COVID-19 disease severity (e.g., kidney dysfunction, hypoxia, multiple organ dysfunction). A decision tree generated by machine learning identified sPLA2-IIA levels as a central node in the stratification of patients who died from COVID-19. Random forest analysis and least absolute shrinkage and selection operator-based (LASSO-based) regression analysis additionally identified sPLA2-IIA and blood urea nitrogen (BUN) as the key variables among 80 clinical indices in predicting COVID-19 mortality. The combined PLA-BUN index performed significantly better than did either one alone. An independent cohort (n = 154) confirmed higher plasma sPLA2-IIA levels in deceased patients compared with levels in plasma from patients with severe or mild COVID-19, with the PLA-BUN index-based decision tree satisfactorily stratifying patients with mild, severe, or fatal COVID-19. With clinically tested inhibitors available, this study identifies sPLA2-IIA as a therapeutic target to reduce COVID-19 mortality.
• Trayssac, M., Clarke, C. J., Stith, J. L., Snider, J. M., Newen, N., Gault, C. R., Hannun, Y. A., & Obeid, L. M. (2021). Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides. Cell death & disease, 12(1), 27.
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  Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence-as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21 (p21) expression, and senescence-associated β-galactosidase activity (SA-β-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-β-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.
• Choi, S., Snider, J. M., Cariello, C. P., Lambert, J. M., Anderson, A. K., Cowart, L. A., & Snider, A. J. (2020). Sphingosine kinase 1 is required for myristate-induced TNFα expression in intestinal epithelial cells. Prostaglandins & other lipid mediators, 149, 106423.
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  Saturated fatty acids (SFA) have been known to trigger inflammatory signaling in metabolic tissues; however, the effects of specific SFAs in the intestinal epithelium have not been well studied. Several previous studies have implicated disruptions in sphingolipid metabolism by oversupply of SFAs in inflammatory process. Also, our previous studies have implicated sphingosine kinase 1 (SK1) and its product sphingosine-1-phosphate (S1P) as having key roles in the regulation of inflammatory processes in the intestinal epithelium. Therefore, to define the role for specific SFAs in inflammatory responses in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, significantly induced the pro-inflammatory cytokine tumor necrosis factor α (TNFα), and it was SK1-dependent. Interestingly, myristate-induced TNFα expression was not suppressed by inhibition of S1P receptors (S1PRs), hinting at a potential novel intracellular target of S1P. Additionally, myristate regulated the expression of TNFα via JNK activation in an SK1-dependent manner, suggesting a novel S1PR-independent target as a mediator between SK1 and JNK in response to myristate. Lastly, a myristate-enriched milk fat-based diet (MFBD) increased expression of TNFα in colon tissues and elevated the S1P to sphingosine ratio, demonstrating the potential of myristate-involved pathobiologies in intestinal tissues. Taken together our studies suggest that myristate regulates the expression of TNFα in the intestinal epithelium via regulation of SK1 and JNK.
• Matmati, N., Hassan, B. H., Ren, J., Shamssedine, A. A., Jeong, E., Shariff, B., Snider, J., Rødkær, S. V., Chen, G., Mohanty, B. K., Zheng, W. J., Obeid, L. M., Røssel-Larsen, M., Færgeman, N. J., & Hannun, Y. A. (2020). Yeast Sphingolipid Phospholipase Gene Regulates the Spindle Checkpoint by a -Dependent Mechanism. Molecular and cellular biology, 40(12).
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  Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase , we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (, , , , and ) as synthetically lethal with The second group, to which belongs, comprises genes involved in the spindle checkpoint (, , , and ), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of Reciprocally, deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to deletion, deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to -mediated nuclear functions.
• Snider, A. J., Seeds, M. C., Johnstone, L., Snider, J. M., Hallmark, B., Dutta, R., Moraga Franco, C., Parks, J. S., Bensen, J. T., Broeckling, C. D., Mohler, J. L., Smith, G. J., Fontham, E. T., Lin, H. K., Bresette, W., Sergeant, S., & Chilton, F. H. (2020). Identification of Plasma Glycosphingolipids as Potential Biomarkers for Prostate Cancer (PCa) Status. Biomolecules, 10(10).
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  Prostate cancer (PCa) is the most common male cancer and the second leading cause of cancer death in United States men. Controversy continues over the effectiveness of prostate-specific antigen (PSA) for distinguishing aggressive from indolent PCa. There is a critical need for more specific and sensitive biomarkers to detect and distinguish low- versus high-risk PCa cases. Discovery metabolomics were performed utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) on plasma samples from 159 men with treatment naïve prostate cancer participating in the North Carolina-Louisiana PCa Project to determine if there were metabolites associated with aggressive PCa. Thirty-five identifiable plasma small molecules were associated with PCa aggressiveness, 15 of which were sphingolipids; nine common molecules were present in both African-American and European-American men. The molecules most associated with PCa aggressiveness were glycosphingolipids; levels of trihexosylceramide and tetrahexosylceramide were most closely associated with high-aggressive PCa. The Cancer Genome Atlas was queried to determine gene alterations within glycosphingolipid metabolism that are associated with PCa and other cancers. Genes that encode enzymes associated with the metabolism of glycosphingolipids were altered in 12% of PCa and >30% of lung, uterine, and ovarian cancers. These data suggest that the identified plasma (glyco)sphingolipids should be further validated for their association with aggressive PCa, suggesting that specific sphingolipids may be included in a diagnostic signature for PCa.
• Sakamoto, W., Canals, D., Salamone, S., Allopenna, J., Clarke, C. J., Snider, J., Obeid, L. M., & Hannun, Y. A. (2019). Probing compartment-specific sphingolipids with targeted bacterial sphingomyelinases and ceramidases. Journal of lipid research, 60(11), 1841-1850.
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  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.
• Schwartz, N. U., Mileva, I., Gurevich, M., Snider, J., Hannun, Y. A., & Obeid, L. M. (2019). Quantifying 1-deoxydihydroceramides and 1-deoxyceramides in mouse nervous system tissue. Prostaglandins & other lipid mediators, 141, 40-48.
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  Accumulation of deoxysphingolipids (deoxySLs) has been implicated in many neural diseases, although mechanisms remain unclear. A major obstacle limiting understanding of deoxySLs has been the lack of a method easily defining measurement of deoxydihydroceramide (deoxydhCer) and deoxyceramide (deoxyCer) in neural tissues. Furthermore, it is poorly understood if deoxySLs accumulate in the nervous system with aging. To facilitate investigation of deoxydhCer and deoxyCer in nervous system tissue, we developed a method to evaluate levels of these lipids in mouse brain, spinal cord, and sciatic nerve. Many deoxydhCers and brain C24-deoxyCer were present at 1, 3, and 6 months of age. Furthermore, while ceramide levels decreased with age, deoxydhCers increased in sciatic nerve and spinal cord, suggesting they may accumulate in peripheral nerves. C22-deoxydhCer was the highest deoxydhCer species in all tissues, suggesting it may be important physiologically. The development of this method will facilitate straightforward profiling of deoxydhCers and deoxyCers and the study of their metabolism and function. These results also reveal that deoxydhCers accumulate in peripheral nerves with normal aging.
• Snider, J. M., Luberto, C., & Hannun, Y. A. (2019). Approaches for probing and evaluating mammalian sphingolipid metabolism. Analytical biochemistry, 575, 70-86.
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  Sphingolipid metabolism plays a critical role in regulating processes that control cellular fate. This dynamic pathway can generate and degrade the central players: ceramide, sphingosine and sphingosine-1-phosphate in almost any membrane in the cell, adding an unexpected level of complexity in deciphering signaling events. While in vitro assays have been developed for most enzymes in SL metabolism, these assays are setup for optimal activity conditions and can fail to take into account regulatory components such as compartmentalization, substrate limitations, and binding partners that can affect cellular enzymatic activity. Therefore, many in-cell assays have been developed to derive results that are authentic to the cellular situation which may give context to alteration in SL mass. This review will discuss approaches for utilizing probes for mammalian in-cell assays to interrogate most enzymatic steps central to SL metabolism. The use of inhibitors in conjunction with these probes can verify the specificity of cellular assays as well as provide valuable insight into flux in the SL network. The use of inhibitors specific to each of the central sphingolipid enzymes are also discussed to assist researchers in further interrogation of these pathways.
• Snider, J. M., Trayssac, M., Clarke, C. J., Schwartz, N., Snider, A. J., Obeid, L. M., Luberto, C., & Hannun, Y. A. (2019). Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis. Journal of lipid research, 60(4), 819-831.
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  Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.
• Snider, J., Wang, D., Bogenhagen, D. F., & Haley, J. D. (2019). Pulse SILAC Approaches to the Measurement of Cellular Dynamics. Advances in experimental medicine and biology, 1140, 575-583.
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  The global measurement of assembly and turnover of protein containing complexes within cells has advanced with the development of pulse stable isotope labelled amino acid approaches. Stable isotope labeling with amino acids in cell culture (SILAC) allows the incorporation of "light" 12-carbon amino acids or "heavy" 13-carbon amino acids into cells or organisms and the quantitation of proteins and peptides containing these amino acid tags using mass spectrometry. The use of these labels in pulse or pulse-chase scenarios has enabled measurements of macromolecular dynamics in cells, on time scales of several hours. Here we review advances with this approach and alternative or parallel strategies. We also examine the statistical considerations impacting datasets detailing mitochondrial assembly, to highlight key parameters in establishing significance and reproducibility.
• Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., & Snider, A. J. (2018). Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 32(10), 5724-5736.
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  Saturated fatty acids (SFAs) have been shown to induce endoplasmic reticulum (ER) stress and chronic inflammatory responses, as well as alter sphingolipid metabolism. Disruptions in ER stress and sphingolipid metabolism have also been implicated in intestinal inflammation. Therefore, to elucidate the roles of SFAs in ER stress and inflammation in intestinal epithelial cells, we examined myristate (C14:0) and palmitate (C16:0). Myristate, but not palmitate, induced ER stress signaling, including activation of inositol-requiring enzyme 1 (IRE1) and X-box binding protein 1 (XBP1) signaling. Myristate significantly increased C14-ceramide levels, whereas palmitate increased several long-chain ceramides. To define the role of ceramide synthases (CerSs) in myristate-induced ER stress, we used the pharmacologic inhibitor, fumonisin B1 (FB1), and small interfering RNA (siRNA) for CerS5 and 6, the primary isoforms that are involved in C14-ceramide generation. FB1 and siRNA for CerS5 or 6 suppressed myristate-induced C14-ceramide generation and XBP1 splicing (XBP1s). Moreover, increased XBP1s induced the downstream expression of IL-6 in a CerS5/6-dependent manner. In addition, a myristate-enriched milk fat-based diet, but not a lard-based diet, increased C14-ceramide, XBP1s, and IL-6 expression in vivo. Taken together, our data suggest that myristate modulates ER stress and cytokine production in the intestinal epithelium via CerS5/6 and C14-ceramide generation.-Choi, S., Snider, J. M., Olakkengil, N., Lambert, J. M., Anderson, A. K., Ross-Evans, J. S., Cowart, L. A., Snider, A. J. Myristate-induced endoplasmic reticulum stress requires ceramide synthases 5/6 and generation of C14-ceramide in intestinal epithelial cells.
• Newcomb, B., Rhein, C., Mileva, I., Ahmad, R., Clarke, C. J., Snider, J., Obeid, L. M., & Hannun, Y. A. (2018). Identification of an acid sphingomyelinase ceramide kinase pathway in the regulation of the chemokine CCL5. Journal of lipid research, 59(7), 1219-1229.
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  Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to produce the biologically active lipid ceramide. Previous studies have implicated ASM in the induction of the chemokine CCL5 in response to TNF-α however, the lipid mediator of this effect was not established. In the present study, we identified a novel pathway connecting ASM and ceramide kinase (CERK). The results show that TNF-α induces the formation of ceramide 1-phosphate (C-1-P) in a CERK-dependent manner. Silencing of CERK blocks CCL5 production in response to TNF-α. Interestingly, cells lacking ASM have decreased C-1-P production following TNF-α treatment, suggesting that ASM may be acting upstream of CERK. Functionally, ASM and CERK induce a highly concordant program of cytokine production and both are required for migration of breast cancer cells. Taken together, these data suggest ASM can produce ceramide which is then converted to C-1-P by CERK, and that C-1-P is required for production of CCL5 and several cytokines and chemokines, with roles in cell migration. These results highlight the diversity in action of ASM through more than one bioactive sphingolipid.
• Rego, A., Cooper, K. F., Snider, J., Hannun, Y. A., Costa, V., Côrte-Real, M., & Chaves, S. R. (2018). Acetic acid induces Sch9p-dependent translocation of Isc1p from the endoplasmic reticulum into mitochondria. Biochimica et biophysica acta. Molecular and cell biology of lipids, 1863(6), 576-583.
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  Changes in sphingolipid metabolism have been linked to modulation of cell fate in both yeast and mammalian cells. We previously assessed the role of sphingolipids in cell death regulation using a well characterized yeast model of acetic acid-induced regulated cell death, finding that Isc1p, inositol phosphosphingolipid phospholipase C, plays a pro-death role in this process. Indeed, isc1∆ mutants exhibited a higher resistance to acetic acid associated with reduced mitochondrial alterations. Here, we show that Isc1p is regulated by Sch9p under acetic acid stress, since both single and double mutants lacking Isc1p or/and Sch9p have the same resistant phenotype, and SCH9 deletion leads to a higher retention of Isc1p in the endoplasmic reticulum upon acetic acid exposure. We also found that the higher resistance of all mutants correlates with higher levels of endogenous mitochondrial phosphorylated long chain bases (LCBPs), suggesting that changing the sphingolipid balance in favour of LCBPs in mitochondria results in increased survival to acetic acid. In conclusion, our results suggest that Sch9p pathways modulate acetic acid-induced cell death, through the regulation of Isc1p cellular distribution, thus affecting the sphingolipid balance that regulates cell fate.
• Ren, J., Saied, E. M., Zhong, A., Snider, J., Ruiz, C., Arenz, C., Obeid, L. M., Girnun, G. D., & Hannun, Y. A. (2018). Tsc3 regulates SPT amino acid choice in by promoting alanine in the sphingolipid pathway. Journal of lipid research, 59(11), 2126-2139.
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  The generation of most sphingolipids (SPLs) starts with condensation between serine and an activated long-chain fatty acid catalyzed by serine palmitoyltransferase (SPT). SPT can also use other amino acids to generate small quantities of noncanonical SPLs. The balance between serine-derived and noncanonical SPLs is pivotal; for example, hereditary sensory and autonomic neuropathy type I results from SPT mutations that cause an abnormal accumulation of alanine-derived SPLs. The regulatory mechanism for SPT amino acid selectivity and physiological functions of noncanonical SPLs are unknown. We investigated SPT selection of amino acid substrates by measuring condensation products of serine and alanine in yeast cultures and SPT use of serine and alanine in a knockout model. We identified the Tsc3 subunit of SPT as a regulator of amino acid substrate selectivity by demonstrating its primary function in promoting alanine utilization by SPT and confirmed its requirement for the inhibitory effect of alanine on SPT utilization of serine. Moreover, we observed downstream metabolic consequences to Tsc3 loss: serine influx into the SPL biosynthesis pathway increased through Ypk1-depenedent activation of SPT and ceramide synthases. This Ypk1-dependent activation of serine influx after Tsc3 knockout suggests a potential function for deoxy-sphingoid bases in modulating Ypk1 signaling.
• Ren, J., Snider, J., Airola, M. V., Zhong, A., Rana, N. A., Obeid, L. M., & Hannun, Y. A. (2018). Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast. Journal of lipid research, 59(1), 162-170.
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  Serine palmitoyltransferase (SPT) catalyzes the rate-limiting step of condensation of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (3KDS). Here, we report a HPLC-ESI-MS/MS method to directly quantify 3KDS generated by SPT. With this technique, we were able to detect 3KDS at a level comparable to that of dihydrosphingosine in yeast An in vitro SPT assay measuring the incorporation of deuterated serine into deuterated 3KDS was developed. The results show that SPT kinetics in response to palmitoyl-CoA fit into an allosteric sigmoidal model, suggesting the existence of more than one palmitoyl-CoA binding site on yeast SPT and positive cooperativity between them. Myriocin inhibition of yeast SPT activity was also investigated and we report here, for the first time, an estimated myriocin for yeast SPT of approximately 10 nM. Lastly, we investigated the fate of serine α-proton during SPT reaction. We provide additional evidence to support the proposed mechanism of SPT catalytic activity in regard to proton exchange between the intermediate NH base formed on the active Lys residue with surrounding water. These findings establish the current method as a powerful tool with significant resolution and quantitative power to study SPT activity.
• Snider, J. M., Snider, A. J., Obeid, L. M., Luberto, C., & Hannun, Y. A. (2018). Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry. Journal of lipid research, 59(6), 1046-1057.
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  Sphingolipids constitute a dynamic metabolic network that interconnects several bioactive molecules, including ceramide (Cer), sphingosine (Sph), Sph 1-phosphate, and Cer 1-phosphate. The interconversion of these metabolites is controlled by a cohort of at least 40 enzymes, many of which respond to endogenous or exogenous stimuli. Typical probing of the sphingolipid pathway relies on sphingolipid mass levels or determination of the activity of individual enzymes. Either approach is unable to provide a complete analysis of flux through sphingolipid metabolism, which, given the interconnectivity of the sphingolipid pathway, is critical information to identify nodes of regulation. Here, we present a one-step in situ assay that comprehensively probes the flux through de novo sphingolipid synthesis, post serine palmitoyltransferase, by monitoring the incorporation and metabolism of the 17 carbon dihydrosphingosine precursor with LC/MS. Pulse labeling and analysis of precursor metabolism identified sequential well-defined phases of sphingolipid synthesis, corresponding to the activity of different enzymes in the pathway, further confirmed by the use of specific inhibitors and modulators of sphingolipid metabolism. This work establishes precursor pulse labeling as a practical tool for comprehensively studying metabolic flux through de novo sphingolipid synthesis and complex sphingolipid generation.
• Edvardson, S., Yi, J. K., Jalas, C., Xu, R., Webb, B. D., Snider, J., Fedick, A., Kleinman, E., Treff, N. R., Mao, C., & Elpeleg, O. (2016). Deficiency of the alkaline ceramidase ACER3 manifests in early childhood by progressive leukodystrophy. Journal of medical genetics, 53(6), 389-96.
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  Leukodystrophies due to abnormal production of myelin cause extensive morbidity in early life; their genetic background is still largely unknown. We aimed at reaching a molecular diagnosis in Ashkenazi-Jewish patients who suffered from developmental regression at 6-13 months, leukodystrophy and peripheral neuropathy.
• Yi, J. K., Xu, R., Jeong, E., Mileva, I., Truman, J. P., Lin, C. L., Wang, K., Snider, J., Wen, S., Obeid, L. M., Hannun, Y. A., & Mao, C. (2016). Aging-related elevation of sphingoid bases shortens yeast chronological life span by compromising mitochondrial function. Oncotarget, 7(16), 21124-44.
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  Sphingoid bases (SBs) as bioactive sphingolipids, have been implicated in aging in yeast. However, we know neither how SBs are regulated during yeast aging nor how they, in turn, regulate it. Herein, we demonstrate that the yeast alkaline ceramidases (YPC1 and YDC1) and SB kinases (LCB4 and LCB5) cooperate in regulating SBs during the aging process and that SBs shortens chronological life span (CLS) by compromising mitochondrial functions. With a lipidomics approach, we found that SBs were increased in a time-dependent manner during yeast aging. We also demonstrated that among the enzymes known for being responsible for the metabolism of SBs, YPC1 was upregulated whereas LCB4/5 were downregulated in the course of aging. This inverse regulation of YPC1 and LCB4/5 led to the aging-related upregulation of SBs in yeast and a reduction in CLS. With the proteomics-based approach (SILAC), we revealed that increased SBs altered the levels of proteins related to mitochondria. Further mechanistic studies demonstrated that increased SBs inhibited mitochondrial fusion and caused fragmentation, resulting in decreases in mtDNA copy numbers, ATP levels, mitochondrial membrane potentials, and oxygen consumption. Taken together, these results suggest that increased SBs mediate the aging process by impairing mitochondrial structural integrity and functions.
• Rajagopalan, V., Canals, D., Luberto, C., Snider, J., Voelkel-Johnson, C., Obeid, L. M., & Hannun, Y. A. (2015). Critical determinants of mitochondria-associated neutral sphingomyelinase (MA-nSMase) for mitochondrial localization. Biochimica et biophysica acta, 1850(4), 628-39.
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  A novel murine mitochondria-associated neutral sphingomyelinase (MA-nSMase) has been recently cloned and partially characterized. The subcellular localization of the enzyme was found to be predominant in mitochondria. In this work, the determinants of mitochondrial localization and its topology were investigated.
• Airola, M. V., Tumolo, J. M., Snider, J., & Hannun, Y. A. (2014). Identification and biochemical characterization of an acid sphingomyelinase-like protein from the bacterial plant pathogen Ralstonia solanacearum that hydrolyzes ATP to AMP but not sphingomyelin to ceramide. PloS one, 9(8), e105830.
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  Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases.
• Geng, T., Hu, W., Broadwater, M. H., Snider, J. M., Bielawski, J., Russo, S. B., Schwacke, J. H., Ross, J., & Cowart, L. A. (2013). Fatty acids differentially regulate insulin resistance through endoplasm reticulum stress-mediated induction of tribbles homologue 3: a potential link between dietary fat composition and the pathophysiological outcomes of obesity. Diabetologia, 56(9), 2078-87.
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  Previous studies have shown that saturated fatty acids cause insulin resistance (IR) that is prevented by unsaturated fatty acids. Tribbles homologue 3 (TRIB3) is a putative endogenous inhibitor of insulin signalling, but its role in insulin signalling is controversial. This study aimed to determine whether fatty acids regulate IR via TRIB3.

Presentations

• Snider, A. J., Snider, J., Doll, C., & Marron, M. (2023, April). Myristate-induced sphingolipid dysregulation results in endoplasmic reticulum stress in intestinal epithelial cells. International Ceramide Conference. Charleston, SC: International Ceramide Conference.
• Snider, A. J., Snider, J., Espinoza, K., Hermanson, K., Beard, C., Schwartz, N., Low, B., Wiles, M., Hannun, Y., & Obeid, L. (2023, April). Characterizing the Role of Sphingolipids in a Novel Mouse Model for Charcot-Marie-Tooth Disease. International Ceramide Conference. Charleston, SC: International Ceramide Conference.

Poster Presentations

• Snider, A. J., Snider, J., Marron, M., Doll, C., & Dahl, B. (2023, June). Myristate-induced sphingolipid dysregulation results in endoplasmic reticulum stress in intestinal epithelial cells. The Intestinal Lipid Metabolism Conference: In Health and Disease. Steamboat Springs, CO: FASEB.
• Warfel, N. A., Warfel, N. A., Chauhan, S. C., Chauhan, S. C., Snider, J., Snider, J., Snider, A. J., Snider, A. J., Cress, A. E., & Cress, A. E. (2022, winter).

  PIM1 derives lipid droplet accumulation via regulation of GSK3β-PPARα signaling axis to promote proliferation and survival in prostate cancer

  . FASEB lipid droplet conferenceFASEB.