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- Title
Controlled Overexpression of Uncoupling Protein 2 in INS-1 Cells Does Not Favorably Affect Parameters of Oxidative Stress But Increases Susceptibility to Damage by Further Uncoupling.
- Authors
Grill, Valdemar; Ogata, Hirotaka; Pettersen, Elin; Jørgensen, Ingrid; Skorpen, Frank; Ma, Zuheng; Ekeberg, Kjartan; Björklund, Anneli
- Abstract
Uncoupling protein 2 (UCP-2) in beta cells is shown to have diabetogenic influence but has also been proposed to exert a beneficial role, coupled to protection against radical oxygen species (ROS), due to mild uncoupling of mitochondria. We investigated this proposition in INS-1 cells, which were transfected to obtain a tet-on, UCP-2 inducible cell line. Induction by 0.1 or 1.0 doxycyclin (dox) µg/ml for 48h increased UCP-2 protein by respectively 178±29 and 424±113 %, n=3. Flow cytometry was used for measuring mitochondrial membrane potential (MMP) by rhodamine 123, oxidative stress by CM-H2DCFDA and apoptosis by annexin V. Cell viability was measured by the MTT assay. Induction by dox (1.0 µg/ml) lowered MMP modestly (by 5.1±2.9%, n=4, p<0.02). In non-induced cells 20 min exposure to 40 µM H202 increased fluorescence by DCF 3.2-fold. The effect of 400 µM H202 was 7.2 fold. Dox induction did not change basal DCF fluorescence (-5.3% of control after induction with 0.1, and +1.0% of control after 1µg/ml of dox). Neither were H202-stimulated responses significantly affected (-6.5% of control response with 40 µM H202 after induction by 0.1 and -7.0 % after induction by 1 µg dox/ml, p<0.4 or more, n=6; similarly no effect at 400 µM H202, n=6). Induction by dox (1 µg/ml) failed to affect the 2.3 fold increase in apoptosis (annexinV positive cells) by camptotecin (added effect by dox +4%, n=3, NS). Exposure to a low concentration (1 µM) of the uncoupling agent FCCP did not decrease cell viability in non-induced cells but decreased it by 19.4±4.0% in dox (0.1 microg/ml)induced cells, n=4, p< 0.02. We conclude that uncoupling by over expressing UCP.-2 fails to positively affect parameters of oxidative stress, whereas it increases susceptibility to negative effects of further uncoupling. A putative protective effect against oxidative stress may be restricted to a permissive one, exerted already at normal levels of UCP-2.
- Subjects
PROTEINS; CELLS; OXIDATIVE stress; PANCREATIC beta cells; APOPTOSIS
- Publication
Diabetes, 2007, Vol 56, pA686
- ISSN
0012-1797
- Publication type
Article