ABSTRACT
Mitochondrial dysfunction has been associated with insulin resistance and diabetes. Decreased mitochondrial density and mitochondrial copy numbers have been found in insulin-resistant individuals. Restoration of the number of mitochondria and normal mitochondrial function has become an important therapeutic target of diabetes. Salicylate, the main active ingredient in aspirin, has been in medicinal use since ancient times. Little information regarding the effects of salicylate on mitochondrial function has been reported. In this study, we assessed the effects of salicylate on the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) signaling pathway and mitochondrial biogenesis in pre-adipocytes. Our findings demonstrate that treatment with salicylate promoted the expression of PGC-1α and its downstream targets nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM). Importantly, salicylate treatment significantly increased the number of mDNA, citrate synthase activity, expression of respiratory chain complex I, and mitochondrial mass, which were suppressed by the specific AMPK inhibitor Compound C. Indeed, salicylate treatment induced the phosphorylation of AMPK, which was involved in the induction of PGC-1α, NRF1, and TFAM. Importantly, inhibition of PGC-1α expression using PGC-1α small RNA interference abolished the effects of salicylate on mitochondrial biogenesis. These results suggest that salicylate has a potential therapeutic capacity against mitochondrial dysfunction in diabetes.
Subject(s)
Adipocytes/drug effects , Hypoglycemic Agents/pharmacology , Mitochondria/drug effects , Organelle Biogenesis , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Salicylic Acid/pharmacology , 3T3-L1 Cells , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Adipocytes/cytology , Adipocytes/metabolism , Animals , Cell Differentiation/drug effects , Citrate (si)-Synthase/genetics , Citrate (si)-Synthase/metabolism , DNA, Mitochondrial/genetics , DNA, Mitochondrial/metabolism , DNA-Binding Proteins/agonists , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Gene Expression Regulation , High Mobility Group Proteins/agonists , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Mice , Mitochondria/genetics , Mitochondria/metabolism , Nuclear Respiratory Factor 1/agonists , Nuclear Respiratory Factor 1/genetics , Nuclear Respiratory Factor 1/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/agonists , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/antagonists & inhibitors , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal TransductionABSTRACT
AIMS: This work explored the effects of irisin on metabolism, gene expression and mitochondrial content in cultured myocytes. METHODS: C2C12 myocytes were treated with various concentrations of irisin for various durations. Glycolysis and oxidative metabolism were quantified by measurement of extracellular acidification and oxygen consumption, respectively. Metabolic gene expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and mitochondrial content was assessed by flow cytometry and confocal microscopy. RESULTS: Cells treated with irisin exhibited significantly increased oxidative metabolism. Irisin treatment also significantly increased mitochondrial uncoupling at various doses and durations. Lastly, treatment with irisin also significantly elevated metabolic gene expression including peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), irisin, glucose transporter 4 (GLUT4) and mitochondrial uncoupling protein 3 (UCP3) leading to increased mitochondrial biogenesis. CONCLUSIONS: Our observations are the first to document increased metabolism in myocytes through irisin-mediated induction of mitochondrial biogenesis and uncoupling with corresponding gene expression. These observations support the need for further investigation into the therapeutic and pharmacological effects of irisin, as well as development of irisin-based therapy.
Subject(s)
Fibronectins/pharmacology , Gene Expression Regulation/drug effects , Glycolysis/drug effects , Mitochondria, Muscle/drug effects , Muscle Fibers, Skeletal/drug effects , Muscle Proteins/metabolism , Oxidative Phosphorylation/drug effects , Animals , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA-Binding Proteins/agonists , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fibronectins/agonists , Fibronectins/genetics , Fibronectins/metabolism , High Mobility Group Proteins/agonists , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Humans , Kinetics , Mice , Mitochondria, Muscle/metabolism , Mitochondrial Turnover/drug effects , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/agonists , Muscle Proteins/genetics , Nuclear Respiratory Factor 1/agonists , Nuclear Respiratory Factor 1/genetics , Nuclear Respiratory Factor 1/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Recombinant Proteins/pharmacology , Transcription Factors/agonists , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Mitochondrial dysfunction contributing to the pathogenesis of glaucomatous neurodegeneration has stimulated considerable interest recently. In this study, we explored the role of peroxisome proliferator activated receptor-γ co-activator 1α (PGC-1α) in resveratrol-triggered mitochondrial biogenesis for preventing apoptosis in a retinal ganglion cell line RGC-5. Our results showed that serum deprivation induced cell apoptosis in a time-dependent manner. Applying resveratrol maintained the normal mitochondrial membrane potential, decreased the levels of both total and cleaved caspase-3, and inhibited the release of cytochrome c, which subsequently enhanced cell survival. Moreover, resveratrol stimulated mitochondrial biogenesis by increasing the absolute quantity of mitochondria as well as their DNA copies. Treatment with resveratrol promoted the protein expression of SIRT1, but not PGC-1α; instead, resveratrol facilitated PGC-1α translocation from the cytoplasm to the nucleus and up-regulated NRF1 and TFAM, which were blocked by nicotinamide. Collectively, we demonstrate that the SIRT1-dependent PGC-1α subcellular translocation following resveratrol application potentially attenuates serum deprivation-elicited RGC-5 cell death, thereby raising the possibility of mitigating glaucomatous retinopathy by enhancement of mitochondrial biogenesis.
