ABSTRACT
Type 2 diabetes is characterized by peripheral insulin resistance and pancreatic beta cell dysfunction. Elevated free fatty acids (FFAs) may impair beta cell function and mass (lipotoxicity). Altered calcium homeostasis may be involved in defective insulin release. The endoplasmic reticulum (ER) is the major intracellular calcium store. Lipotoxicity induces ER stress and in parallel an ER calcium depletion through unknown ER calcium leak channels. The main purposes of this study is first to identify one of these channels and secondly, to check the opportunity to restore beta cells function (i.e., insulin secretion) after pharmacological inhibition of ER calcium store depletion. We investigated the functionality of translocon, an ER calcium leak channel and its involvement on FFAs-induced alterations in MIN6B1 cells and in human pancreatic islets. We evidenced that translocon acts as a functional ER calcium leak channel in human beta cells using anisomycin and puromycin (antibiotics), respectively blocker and opener of this channel. Puromycin induced a significant ER calcium release, inhibited by anisomycin pretreatment. Palmitate treatment was used as FFA model to induce a mild lipotoxic effect: ER calcium content was reduced, ER stress but not apoptosis were induced and glucose induced insulin secretion was decreased in our beta cells. Interestingly, translocon inhibition by chronic anisomycin treatment prevented dysfunctions induced by palmitate, avoiding reticular calcium depletion, ER stress and restoring insulin secretion. Our results provide for the first time compelling evidence that translocon actively participates to the palmitate-induced ER calcium leak and insulin secretion decrease in beta cells. Its inhibition reduces these lipotoxic effects. Taken together, our data indicate that TLC may be a new potential target for the treatment of type 2 diabetes.
Subject(s)
Insulin-Secreting Cells/drug effects , Palmitates/toxicity , Protein Translocation Systems/physiology , Animals , Anisomycin/pharmacology , Apoptosis/drug effects , Calcium/metabolism , Calcium Signaling/drug effects , Caspases/metabolism , Cells, Cultured , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/drug effects , Enzyme Activation/drug effects , Genes, Reporter , Heat-Shock Proteins/genetics , Heat-Shock Proteins/physiology , Homeostasis , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism , Ion Transport/drug effects , Mice , Protein Transport/drug effects , Puromycin/pharmacology , RNA Interference , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
Glycogen synthase kinase-3ß (GSK3ß) is a multifunctional kinase whose inhibition is known to limit myocardial ischemia-reperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia-reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3ß might have a role in the Ca(2+) transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3ß protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3ß specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP3Rs) Ca(2+) channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3ß decreased protein interaction of IP3R with the Ca(2+) channeling complex, impaired SR/ER Ca(2+) release and reduced the histamine-stimulated Ca(2+) exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3ß activity and IP3R phosphorylation, which leads to enhanced transfer of Ca(2+) from SR/ER to mitochondria. Inhibition of GSK3ß at reperfusion reduced both IP3R phosphorylation and SR/ER Ca(2+) release, which consequently diminished both cytosolic and mitochondrial Ca(2+) concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3ß at reperfusion diminishes Ca(2+) leak from IP3R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca(2+) overload and subsequent cell death.
Subject(s)
Calcium Signaling , Glycogen Synthase Kinase 3/physiology , Mitochondria, Heart/metabolism , Myocardial Reperfusion Injury/enzymology , Sarcoplasmic Reticulum/metabolism , Animals , Calcium/metabolism , Cell Line , Glycogen Synthase Kinase 3 beta , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Male , Mice, Inbred C57BL , Myocardial Reperfusion Injury/pathology , Myocardium/enzymology , Myocardium/pathology , Myocytes, Cardiac/enzymology , Phosphorylation , Protein Processing, Post-TranslationalABSTRACT
RATIONALE: How ischemic postconditioning can inhibit opening of the mitochondrial permeability transition pore (PTP) and subsequent cardiac myocytes death at reperfusion remains unknown. Recent studies have suggested that de-acetylation of cyclophilin D (CyPD) by sirtuin 3 (SIRT3) can modulate its binding to the PTP. OBJECTIVE: The aim of the present study was to examine whether ischemic postconditioning (PostC) might activate SIRT3 and consequently prevent lethal myocardial reperfusion injury through a deacetylation of CyPD. METHODS AND RESULTS: Using hypoxia-reoxygenation (H/R) in H9C2 cells, we showed that SIRT3 overexpression prevented CyPD acetylation, limited PTP opening and reduced cell death by 24%. In vitro modification of the CyPD acetylation status in MEFs by site-directed mutagenesis altered capacity of PTP opening by calcium. Calcium Retention Capacity (CRC) was significantly decreased with CyPD-KQ that mimics acetylated protein compared with CyPD WT (871 ± 266 vs 1193 ± 263 nmoles Ca(2+)/mg protein respectively). Cells expressing non-acetylable CyPD mutant (CyPD-KR) displayed 20% decrease in cell death compared to cells expressing CyPD WT after H/R. Correspondingly, in mice we showed that cardiac ischemic postconditioning could not reduce infarct size and CyPD acetylation in SIRT3 KO mice, and was unable to restore CRC in mitochondria as it is observed in WT mice. CONCLUSIONS: Our study suggests that the increased acetylation of CyPD following myocardial ischemia-reperfusion facilitates PTP opening and subsequent cell death. Therefore ischemic postconditioning might prevent lethal reperfusion injury through an increased SIRT3 activity and subsequent attenuation of CyPD acetylation at reperfusion.
Subject(s)
Cyclophilins/metabolism , Ischemic Postconditioning , Myocardial Reperfusion Injury/metabolism , Sirtuin 3/metabolism , Acetylation , Animals , Cell Death , Cell Hypoxia , Peptidyl-Prolyl Isomerase F , Male , Membrane Potential, Mitochondrial/drug effects , Mice, Knockout , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Oxygen/pharmacology , RatsABSTRACT
The myocardial infarction represents a major cause of mortality. The deleterious phenomena arising during the ischaemia and the reperfusion of the myocardium are studied for more than 40 years. We thought for a long time that the ischaemia was the harmful stage, at the origin of the decrease of the energy stores, the dysregulation of the ionic homeostasis and the metabolic deregulation. We know now that the reperfusion itself is also a source of noxious effects (calcium overload, free radicals production, mitochondrion alteration). To combat these deleterious processes, two maneuvers demonstrated their efficiency by protecting the ischemic myocardium : it is the preconditioning and the postconditionning.
