Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic beta-cells.
The phagocytic NADPH oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic beta-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic beta-cells. Incubation of isolated beta-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic beta-cells.
Syed
Ismail
I
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy, Wayne State University, Detroit, MI 48201, United States.
Jayaram
Bhavaani
B
Subasinghe
Wasanthi
W
Kowluru
Anjaneyulu
A
D000818:Animals; D002460:Cell Line; D002518:Ceramides / Q000096:biosynthesis / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism*; D008054:Lipid Peroxides / Q000096:biosynthesis; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D019308:Palmitic Acid / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013481:Superoxides / Q000378:metabolism*; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D020830:rac1 GTP-Binding Protein / Q000502:physiology*
D002518:Ceramides; D020662:Guanine Nucleotide Exchange Factors; D008054:Lipid Peroxides; D009363:Neoplasm Proteins; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C513259:Tiam1 protein, rat; D013481:Superoxides; D019308:Palmitic Acid; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein