Abstract
BackgroundAntioxidant enzymes play a fundamental role in counteracting oxidative stress induced by high glucose. Although mitochondrial superoxide dismutase (SOD2) is the principal defence against the toxicity of superoxide anions, the mechanism of its inactivation in diabetic subjects is still poorly understood. Recently, microRNA-21 has been associated with diabetes, although its function remains unclear. We sought to explore the mechanism underlying defective SOD2 antioxidant response in HUVECs during exposures to constant high glucose and oscillating glucose (as glucose variability model, GV) and the role of miR-21 in increasing the susceptibility to oxidative stress by disrupting reactive oxygen species (ROS) homeostasis.MethodsHUVECs exposed for 1 week to constant high glucose and GV were subjected to quantitative electron paramagnetic resonance for ROS measurements. Superoxide anions, SOD2 protein levels and mitochondrial membrane potential (ΔΨm) were also evaluated. Endogenous miR-21 and its putative ROS-homeostatic target genes (KRIT1, FoxO1, NFE2L2 and SOD2) were tested using mimic-miR-21 and quantified by qPCR. Luciferase assays were performed to test miR-21/3′-UTR-SOD2 binding.ResultsWe observed upregulation of microRNA-21, overproduction of superoxide anions and total ROS generation, depolarisation of the mitochondrial membrane potential (ΔΨm) and defective SOD2 antioxidant response in HUVECs subjected to constant high glucose and GV exposures. We also found that exogenous mimic-microRNA-21 targeted putative microRNA-21 ROS-homeostatic target genes, e.g., KRIT1, NRF2 and SOD2, which were significantly downregulated. All these effects were reverted by a microRNA-21 inhibitor, which improved SOD2 and KRIT1 expression, reduced the levels of ROS production and ameliorated ΔΨm.ConclusionsOur data demonstrate the association of microRNA-21 with oscillating and high glucose and early mitochondrial dysfunction. We found that microRNA-21 may promote the suppression of homeostatic signalling that normally limits ROS damage. These data provide novel clues about the inhibition of microRNA-21 as a new therapeutic approach to protect against cellular oxidative injury in glucose variability and diabetes.
Highlights
Type 2 diabetes (T2D) is a complex metabolic disease associated with insulin resistance, obesity and the development of cardiovascular disorders, whose central factors are involved in the occurrence of high and prolonged cellular oxidative stress (Ox-S) [1]
We aimed to demonstrate that glycaemic variability (GV) and constant high glucose induce the downregulation of a reactive oxygen species (ROS) homeostasis regulator, Krev/Rap1 interaction trapped-1 (KRIT1), and the defective superoxide dismutase 2 (SOD2) antioxidant response via miR-21 activation
Intracellular miR‐21 is induced by oscillating (OG) and high glucose (HG), affecting ROS generation In primary human umbilical vein endothelial cells (HUVECs), in both oscillating glucose (OG) and HG conditions, miR-21 expression levels were increased with respect to controls (p < 0.05 OG vs normal glucose (NG), p < 0.01 HG vs NG) (Fig. 1a)
Summary
Type 2 diabetes (T2D) is a complex metabolic disease associated with insulin resistance, obesity and the development of cardiovascular disorders (micro- and macrovascular), whose central factors are involved in the occurrence of high and prolonged cellular oxidative stress (Ox-S) [1]. In our previous in vitro studies, we showed that GV may have more deleterious effects on endothelial cells than high glucose and that it may activate several metabolic pathways directly responsible for cellular damage [7, 8]. The mechanisms responsible for endothelial damage are closely related to hyperglycaemiainduced mitochondrial reactive oxygen species (ROS) overproduction, resulting in defective ROS homeostasis and inactivation of antioxidant responses, which are responsible for controlling the rate of radicals produced under stress conditions. We sought to explore the mechanism under‐ lying defective SOD2 antioxidant response in HUVECs during exposures to constant high glucose and oscillating glucose (as glucose variability model, GV) and the role of miR-21 in increasing the susceptibility to oxidative stress by disrupting reactive oxygen species (ROS) homeostasis
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