Jaburek 2014 Abstract MiP2014
|The role of phospholipase A2γ in the regulation of mitochondrial uncoupling protein 2–dependent antioxidant function.|
Mitochondrial uncoupling protein 2 (UCP2) has been suggested to participate in the attenuation of the reactive oxygen species production, but the mechanism of action and the physiological significance of UCP2 activity remain controversial. The protonophoretic function of recombinant reconstituted UCP2 is essentially dependent on non-esterified fatty acids , and we showed that mitochondrial phospholipase A2γ participates in the regulation of UCP2 function [2,3].
Because UCP2 plays an antioxidant role in pancreatic β–cells , we also tested our hypothesis of iPLA2γ–dependent regulation of UCP2, using the model of INS–1E insulinoma cells. High-resolution respirometry and parallel fluorometric detection of membrane potential and mitochondrial superoxide formation revealed pro-oxidant–induced increase in respiration, decrease in mitochondrial membrane potential and decrease in mitochondrial superoxide formation in non-targeting shRNA INS–1E controls (ntgINS–1E) but not in UCP2–silenced and iPLA2γ–silenced cells. In addition, we observed identical glucose–stimulated insulin secretion in ntgINS–1E controls, UCP2–silenced and iPLA2γ–silenced cells in the absence of a pro-oxidant insult. Addition of the pro-oxidant tert-butyl hydroperoxide resulted in markedly elevated insulin release in both UCP2–silenced and iPLA2γ–silenced cells but not in ntgINS–1E controls.
Fatty acids are important for normal function of pancreatic β-cells, but elevated levels of free fatty acids are associated with increased production of reactive oxygen species and compromised glucose-stimulated insulin secretion . Therefore, we tested whether the UCP2–mediated, iPLA2γ–dependent antioxidant action protects pancreatic β–cells from acute cytotoxic effects of saturated fatty acids. We exposed the INS-1E insulinoma cells to various concentrations of palmitate and measured the kinetics of insulin secretion and the rate of superoxide production in the mitochondrial matrix. Physiologically relevant concentrations of palmitate (10–30 nmol∙10-6 cells) caused elevated insulin secretion in ntgINS–1E controls but markedly inhibited insulin secretion in both UCP2–silenced and iPLA2γ–silenced cells. Corresponding concentrations of palmitate also led to attenuation of mitochondrial superoxide formation in ntgINS–1E controls but not in UCP2–silenced or iPLA2γ–silenced cells.
These results contribute to the understanding of UCP2–dependent regulation of mitochondrial superoxide production and insulin secretion in pancreatic β-cells and to the understanding of free fatty acid–mediated antioxidant function provided by synergic actions of iPLA2γ and UCP2. Our observations further indicate that UCP2 and iPLA2γ protect β–cells against toxicity associated with acute moderate fatty acid intake.
• O2k-Network Lab: CZ Prague Jezek P
Labels: MiParea: Respiration Pathology: Diabetes Stress:Oxidative stress;RONS
Tissue;cell: Islet cell;pancreas;thymus Preparation: Intact cells Enzyme: Uncoupling protein Regulation: mt-Membrane potential, Fatty acid
HRR: Oxygraph-2k Event: B2, Oral MiP2014
Dep Membrane Transport Biophys, Inst Physiol, Acad Sc Czech Republic, Prague, Czech Republic. - [email protected]
References and acknowledgements
Supported by Grant Agency of the Czech Republic, grant No. P302/10/034, P305/12/1247, and P304/10/P204.
- Ježek P, Jabůrek M, Garlid KD (2010) Channel character of uncoupling protein-mediated transport. FEBS Lett 584: 2135-41.
- Ježek J, Jabůrek M, Zelenka J, Ježek P (2010) Mitochondrial phospholipase A2 activated by reactive oxygen species in heart mitochondria induces mild uncoupling. Physiol Res 59: 737-47.
- Jabůrek M, Ježek J, Zelenka J, Ježek P (2013) Antioxidant activity by a synergy of redox-sensitive mitochondrial phospholipase A2 and uncoupling protein-2 in lung and spleen. Int J Biochem Cell Biol 45: 816-25.
- Ježek P, Olejár T, Smolková K, Ježek J, Dlasková A, Plecitá-Hlavatá L, Zelenka J, Špaček T, Engstová H, Pajuelo Reguera D, Jabůrek M (2014) Antioxidant and regulatory role of mitochondrial uncoupling protein UCP2 in pancreatic beta-cells. Physiol Res 63: 73-91.