Discrete and Complementary Mechanisms of Protection of β-Cells Against Cytokine-Induced and Oxidative Damage Achieved by bcl-2 Overexpression and a Cytokine Selection Strategy
- 1Touchstone Center for Diabetes Research, Department of Biochemistry and Biomedical Engineering Program, University of Texas Southwestern Medical Center, Dallas, Texas
- 2Sarah W. Stedman Nutrition and Metabolism Center, Department of Pharmacology and Cancer Biology, Division of Endocrinology, Metabolism, and Nutrition, Department of Medicine, Duke University Medical Center, Durham, North Carolina
We have been investigating the potential utility of engineered cell lines as surrogates for primary islet cells in treatment of type 1 diabetes. To this end, two strategies that have emerged for procuring cell lines with resistance to immune-mediated damage are 1) selection of cytokine-resistant cell lines by growth of INS-1 insulinoma cells in iteratively increasing concentrations of interleukin (IL)-1β + γ-interferon (IFN-γ), and 2) stable overexpression of the anti-apoptotic gene bcl-2 in INS-1 cells. Herein, we show that bcl-2−overexpressing cells are resistant to the cytotoxic effects of reactive oxygen and nitrogen species (ROS/RNS), but are only modestly protected against high concentrations of IL-1β + INF-γ, whereas the converse is true in cytokine selected cells. We also found that the combination of bcl-2 expression and cytokine selection confers a broader spectrum of resistance than either procedure alone, such that the resultant cells are highly resistant to cytokines and ROS/RNS, with no impairment in glucose-stimulated insulin secretion. INS-1−derived cells with combined bcl-2 expression and cytokine selection are also more resistant to damage induced by coculture with mitogen-activated peripheral blood mononuclear cells. Surprisingly, application of the cytokine selection procedure to bcl-2−overexpressing cells does not result in impairment of nuclear factor-κB translocation, iNOS expression, and NO production, as clearly occurs upon application of the selection procedure to cells without bcl-2 overexpression. Further investigation of the diverse pathways involved in the development of cytokine and ROS/RNS resistance may define simplified and specific strategies for preservation of β-cell mass.
Address correspondence and reprint requests to Christopher B. Newgard, PhD, Department of Pharmacology and Cancer Biology, DUMC 3813, LSRC 351, Duke University Medical Center, Durham, NC 27710. E-mail:.
Received for publication 1 November 2002 and accepted in revised form 28 February 2003.
C.B.N. and H.E.H. are supported by a grant from Takeda Chemicals, Osaka, Japan.
GSIS, glucose-stimulated insulin secretion; IFN-γ, γ-interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MnSOD, manganese superoxide dismutase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; l-NMMA, NG-monomethyl-l-arginine; PBMC, peripheral blood mononuclear cell; PMA, phorbol-12-myristate-13-acetate; PMA/I, PMA + 1 μg/ml ionomycin; PMA/I + LPS, 10 ng/ml PMA + 1 μg/ml ionomycin + 10 μg/ml LPS; RNS, reactive nitrogen species; ROS, reactive oxygen species; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acetylpenicillamine; STAT, signal transducer and activator of transcription; STZ, streptozotocin; TNF-α, tumor necrosis factor-α.