Neutral Ceramidase Secreted Via Exosome Protects Against Palmitate-Induced Apoptosis in INS-1 Cells

Abstract

Aims: To investigate the effects of neutral ceramidase (NCDase) packaged in exosomes that are secreted from β-cells on free fatty acid (FFA)-induced β-cells apoptosis and its role in regulation of sphingolipid-mediated signaling pathway.

Methods: HPLC and Western blotting were performed to determine NCDase activity and expression. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis. Electrospray ionization tandem mass spectrometry was used for ceramide (Cer), sphingosine-1-phosphate (S1P), and sphingosine (SPH) determination.

Results: INS-1 cells over-expressed NCDase secreted active NCDase via exosomes. Exosomes isolated from the cultured medium of INS-1 cells that oxpressed NCDase could ameliorate palmitate-induced apoptosis. Furthermore, the results showed that exosome-derived NCDase treatment reduced intracellular Cer/S1P ratio.

Conclusions: β-cell secreted active NCDase via exosome, the exosome-packaged-NCDase protected β-cells from FFA-induced apoptosis through regulating sphingolipid metabolites and it might be a potential treatment for β-cell lipotoxicity and type 2 diabetes.

Key words

neutral ceramidase - exosome - lipotoxicity - β-cell - type 2 diabetes

^* These authors contribute equally to this work.

• References

• 1 Giacca A, Xiao C, Oprescu AI et al. Lipid-induced pancreatic beta-cell dysfunction: focus on in vivo studies. American journal of physiology Endocrinology and metabolism 2011; 300: E255-E262
• 2 Bikman BT, Summers SA. Ceramides as modulators of cellular and whole-body metabolism. The Journal of clinical investigation 2011; 121: 4222-4230
• 3 Chaurasia B, Summers SA. Ceramides – lipotoxic inducers of metabolic disorders. Trends in endocrinology and metabolism: TEM 2015; 26: 538-550
• 4 Bellini L, Campana M, Mahfouz R et al. Targeting sphingolipid metabolism in the treatment of obesity/type 2 diabetes. Expert opinion on therapeutic targets 2015; 19: 1037-1050
• 5 Manukyan L, Ubhayasekera SJ, Bergquist J et al. Palmitate-induced impairments of beta-cell function are linked with generation of specific ceramide species via acylation of sphingosine. Endocrinology 2015; 156: 802-812
• 6 Veret J, Bellini L, Giussani P et al. Roles of Sphingolipid Metabolism in Pancreatic beta Cell Dysfunction Induced by Lipotoxicity. Journal of clinical medicine 2014; 3: 646-662
• 7 Qi Y, Chen J, Lay A et al. Loss of sphingosine kinase 1 predisposes to the onset of diabetes via promoting pancreatic beta-cell death in diet-induced obese mice. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2013; 27: 4294-4304
• 8 Japtok L, Schmitz EI, Fayyaz S et al. Sphingosine 1-phosphate counteracts insulin signaling in pancreatic beta-cells via the sphingosine 1-phosphate receptor subtype 2. FASEB journal: official publication of the Federation of American Societies for Experimental Biology 2015; 29: 3357-3369
• 9 Saied EM, Arenz C. Small molecule inhibitors of ceramidases. Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology 2014; 34: 197-212
• 10 Zhu Q, Jin JF, Shan XH et al. Chronic activation of neutral ceramidase protects beta-cells against cytokine-induced apoptosis. Acta pharmacologica Sinica 2008; 29: 593-599
• 11 Tani M, Igarashi Y, Ito M. Involvement of neutral ceramidase in ceramide metabolism at the plasma membrane and in extracellular milieu. The Journal of biological chemistry 2005; 280: 36592-36600
• 12 Zhu Q, Kang J, Miao H et al. Low-dose cytokine-induced neutral ceramidase secretion from INS-1 cells via exosomes and its anti-apoptotic effect. The FEBS journal 2014; 281: 2861-2870
• 13 Tkach M, Thery C. Communication by extracellular vesicles: where we are and where we need to go. Cell 2016; 164: 1226-1232
• 14 Zhu Q, Zhong JJ, Jin JF et al. a chemical chaperone, prevents palmitate-induced apoptosis in pancreatic beta-cells by reducing ER stress. Experimental and clinical endocrinology & diabetes: official journal, German Society of Endocrinology [and] German Diabetes Association 2013; 121: 43-47
• 15 Lee J, Yeganeh B, Ermini L et al. Sphingolipids as cell fate regulators in lung development and disease. Apoptosis: an international journal on programmed cell death 2015; 20: 740-757
• 16 Jung JS, Choi MJ, Ko HM et al. Short-chain C2 ceramide induces heme oxygenase-1 expression by upregulating AMPK and MAPK signaling pathways in rat primary astrocytes. Neurochemistry international 2016; 94: 39-47
• 17 Cingolani F, Futerman AH, Casas J. Ceramide synthases in biomedical research. Chemistry and physics of lipids 2016; 197: 25-32
• 18 Xu R, Wang K, Mileva I et al. Alkaline ceramidase 2 and its bioactive product sphingosine are novel regulators of the DNA damage response. Oncotarget 2016; 7: 18440-18457
• 19 Petrache I, Berdyshev EV. Ceramide signaling and metabolism in pathophysiological states of the lung. Annual review of physiology 2016; 78: 463-480
• 20 Jernigan PL, Makley AT, Hoehn RS et al. The role of sphingolipids in endothelial barrier function. Biological chemistry 2015; 396: 681-691
• 21 Tanabe K, Liu Y, Hasan SD et al. Glucose and fatty acids synergize to promote B-cell apoptosis through activation of glycogen synthase kinase 3beta independent of JNK activation. PloS one 2011; 6: e18146
• 22 Ferguson SW, Nguyen J. Exosomes as therapeutics: The implications of molecular composition and exosomal heterogeneity. Journal of controlled release: official journal of the Controlled Release Society 2016; 228: 179-190
• 23 Lawson C, Vicencio JM, Yellon DM et al. Microvesicles and exosomes: new players in metabolic and cardiovascular disease. The Journal of endocrinology 2016; 228: R57-R71
• 24 Zhang HG, Grizzle WE. Exosomes: a novel pathway of local and distant intercellular communication that facilitates the growth and metastasis of neoplastic lesions. The American journal of pathology 2014; 184: 28-41
• 25 Burke M, Choksawangkarn W, Edwards N et al. Exosomes from myeloid-derived suppressor cells carry biologically active proteins. Journal of proteome research 2014; 13: 836-843
• 26 Reiners KS, Dassler J, Coch C et al. Role of Exosomes released by dendritic cells and/or by tumor targets: regulation of NK cell plasticity. Frontiers in immunology 2014; 5: 91
• 27 Ito M, Okino N, Tani M. New insight into the structure, reaction mechanism, and biological functions of neutral ceramidase. Biochimica et biophysica acta 2014; 1841: 682-691
• 28 Inoue T, Okino N, Kakuta Y et al. Mechanistic insights into the hydrolysis and synthesis of ceramide by neutral ceramidase. The Journal of biological chemistry 2009; 284: 9566-9577
• 29 Mao C, Obeid LM. Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate. Biochimica et biophysica acta 2008; 1781: 424-434
• 30 Holland WL, Miller RA, Wang ZV et al. Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin. Nature medicine 2011; 17: 55-63