L- threo -C6-pyridinium-ceramide bromide, a novel cationic ceramide, induces NADPH oxidase activation, mitochondrial dysfunction and loss in cell viability in INS 832/13 β-cells.
BACKGROUND
Emerging evidence indicates that exposure of isolated pancreatic β-cells to elevated glucose [glucotoxicity] or saturated fatty acids such as palmitate [lipotoxicity] or both [glucolipotoxicity] results in excessive intracellular oxidative stress mediated by phagocyte-like NADPH oxidase [Nox2]. Pharmacological evidence also implicates the intracellular generation of ceramide [CER] as one of the mediators of palmitate-induced cytotoxicity of the islet β- cell. Herein, we investigated the effects of L-threo-C6-pyridinium-ceramide bromide, a novel water soluble cationic ceramide [Ws-CER], on mitochondrial function and cell viability in insulin-secreting INS 832/13 cells.


METHODS
Ws-CER, was synthesized as we reported earlier. Rac1 activation was quantitated by pull-down assay. Mitochondrial membrane potential was quantitated by JC-1 staining. Nox2 subunit expression and caspase-3 activity were determined by Western blotting.


RESULTS
Our findings suggested a marked increase in the Nox2 activation [i.e., ROS generation and subunit expression and activation] in cells exposed to Ws-CER. We also noticed a significant reduction in mitochondrial membrane potential, increased in caspase-3 activity and associated loss in cell viability in Ws-CER-treated cells.


CONCLUSIONS
Based on these data we conclude that ceramide-induced Nox2-mediated oxidative stress couples mitochondrial dysfunction to loss in cell viability in the pancreatic β-cell.
Beta-Cell Biochemistry Laboratory, John D. Dingell VA Medical Center and Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201 , USA.
Ismail
I
Syed
Zdzislaw M
ZM
Szulc
Besim
B
Ogretmen
Anjaneyulu
A
Kowluru
D000818:Animals; D001965:Bromides / Q000737:chemistry / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D053078:Membrane Potential, Mitochondrial; D008928:Mitochondria / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D018384:Oxidative Stress; D011726:Pyridinium Compounds / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
C539200:2-N-(6'-(1''-pyridinium)hexanoyl)sphingosine; D001965:Bromides; D002518:Ceramides; D008562:Membrane Glycoproteins; D011726:Pyridinium Compounds; D017382:Reactive Oxygen Species; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D053148:Caspase 3