Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.     

Enhanced autophagy plays a cardinal role in mitochondrial dysfunction in type 2 diabetic Goto-Kakizaki (GK) rats: ameliorating effects of (-)-epigallocatechin-3-gallate

Oxidative stress and mitochondrial dysfunction are known to play important roles in type 2 diabetes mellitus (T2DM) and insulin resistance. However, the pathology of T2DM remains complicated; in particular, the mechanisms of mitochondrial dysfunction in skeletal muscle and other insulin-sensitive tissues are as yet unclear. In the present study, we investigated the underlying mechanisms of oxidative stress and mitochondrial dysfunction by focusing on mitochondrial dynamics, including mitochondrial biogenesis and autophagy, in skeletal muscle of a nonobese diabetic animal model--the Goto-Kakizaki (GK) rat. The results showed that GK rats exhibited impaired glucose metabolism, increased oxidative stress and decreased mitochondrial function. These dysfunctions were found to be associated with induction of LC3B, Beclin1 and DRP1 (key molecules mediating the autophagy pathway), while they appeared not to affect the mitochondrial biogenesis pathway. In addition, (-)-epigallocatechin-3-gallate (EGCG) was tested as a potential autophagy-targeting nutrient, and we found that EGCG treatment improved glucose tolerance and glucose homeostasis in GK rats, and reduced oxidative stress and mitochondrial dysfunction in skeletal muscle. Amelioration of excessive muscle autophagy in GK rats through the down-regulation of the ROS-ERK/JNK-p53 pathway leads to improvement of glucose metabolism, reduction of oxidative stress and inhibition of mitochondrial loss and dysfunction. These results suggest (a) that hyperglycemia-associated oxidative stress may induce autophagy through up-regulation of the ROS-ERK/JNK-p53 pathway, which may contribute to mitochondrial loss in soleus muscle of diabetic GK rats, and (b) that EGCG may be a potential autophagy regulator useful in treatment of insulin resistance.     

Mst1 deletion reduces hyperglycemia-mediated vascular dysfunction via attenuating mitochondrial fission and modulating the JNK signaling pathway

Diabetes is a leading cause of microvascular complications, such as nephropathy and retinopathy. Recent studies have proposed that hyperglycemia-induced endothelial cell dysfunction is modulated by mitochondrial stress. Therefore, our experiment was to detect the upstream mediator of mitochondrial stress in hyperglycemia-treated endothelial cells with a focus on macrophage-stimulating 1 (Mst1) and mitochondrial fission. Our data illuminated that hyperglycemia incubation reduced cell viability, as well as increased apoptosis ratio in endothelial cell, and this alteration seemed to be associated with Mst1 upregulation. Inhibition of Mst1 via transfection of Mst1 siRNA into an endothelial cell could sustain cell viability and maintain mitochondrial function. At the molecular levels, endothelial cell death was accompanied with the activation of mitochondrial oxidative stress, mitochondrial apoptosis, and mitochondrial fission. Genetic ablation of Mst1 could reduce mitochondrial oxidative injury, block mitochondrial apoptosis, and repress mitochondrial fission. Besides, we also found Mst1 triggered mitochondrial dysfunction as well as endothelial cell damage through augmenting JNK pathway. Suppression of JNK largely ameliorated the protective actions of Mst1 silencing on hyperglycemia-treated endothelial cells and sustain mitochondrial function. The present study identifies Mst1 as a primary key mediator for hyperglycemia-induced mitochondrial damage and endothelial cell dysfunction. Increased Mst1 impairs mitochondrial function and activates endothelial cell death via opening mitochondrial death pathway through JNK.     

The Sodium-Glucose Co-Transporter 2 Inhibitor Empagliflozin Improves Diabetes-Induced Vascular Dysfunction in the Streptozotocin Diabetes Rat Model by Interfering with Oxidative Stress and Glucotoxicity

Objective

In diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress.

Methods

Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE).

Results

Treatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy.

Conclusions

Empagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.     

Liquiritin attenuates advanced glycation end products-induced endothelial dysfunction via RAGE/NF-κB pathway in human umbilical vein endothelial cells

Advanced glycation end products (AGEs)-induced vasculopathy, including oxidative stress, inflammation and apoptosis responses, contributes to the high morbidity and mortality of coronary artery diseases in diabetic patients. The present study was conducted to evaluate the protective activity of liquiritin (Liq) on AGEs-induced endothelial dysfunction and explore its underlying mechanisms. After pretreatment with Liq, a significant reduction in AGEs-induced apoptosis, as well as reactive oxygen species generation and malondialdehyde level in human umbilical vein endothelial cells (HUVECs) were observed via acridine orange/ethidium bromide fluorescence staining test. Notably, Liq also significantly increased AGEs-reduced superoxide dismutase activity. Furthermore, the pretreatment with receptor for advanced glycation end products (RAGE)-antibody or Liq remarkably down-regulated TGF-beta1 and RAGE protein expressions and significantly blocked NF-κB activation which were proved by immunocytochemistry or immunofluorescence assays. These results indicated that Liq held potential for the protection on AGEs-induced endothelial dysfunction via RAGE/NF-κB pathway in HUVECs and might be a promising agent for the treatment of vasculopathy in diabetic patients.     

Hyperinsulinemia-induced vascular smooth muscle cell (VSMC) migration and proliferation is mediated by converging mechanisms of mitochondrial dysfunction and oxidative stress

Atherosclerosis is one of the major complications of diabetes and involves endothelial dysfunction, matrix alteration, and most importantly migration and proliferation of vascular smooth muscle cells (VSMCs). Although hyperglycemia and hyperinsulinemia are known to contribute to atherosclerosis, little is known about the specific cellular signaling pathways that mediate the detrimental hyperinsulinemic effects in VSMCs. Therefore, we investigated the cellular mechanisms of hyperinsulinemia-induced migration and proliferation of VSMCs. VSMCs were treated with insulin (100 nM) for 6 days and subjected to various physiological and molecular investigations. VSMCs subjected to hyperinsulinemia exhibited increased migration and proliferation, and this is paralleled by oxidative stress [increased NADPH oxidase activity, NADPH oxidase 1 mRNA expression, and reactive oxygen species (ROS) generation], alterations in mitochondrial physiology (membrane depolarization, decreased mitochondrial mass, and increased mitochondrial ROS), changes in mitochondrial biogenesis-related genes (mitofusin 1, mitofusin 2, dynamin-related protein 1, peroxisome proliferator-activated receptor gamma coactivator 1-alpha, peroxisome proliferator-activated receptor gamma coactivator 1-beta, nuclear respiratory factor 1, and uncoupling protein 2), and increased Akt phosphorylation. Diphenyleneiodonium, a known NADPH oxidase inhibitor significantly inhibited migration and proliferation of VSMCs and normalized all the above physiological and molecular perturbations. This study suggests a plausible crosstalk between mitochondrial dysfunction and oxidative stress under hyperinsulinemia and emphasizes counteracting mitochondrial dysfunction and oxidative stress as a novel therapeutic strategy for atherosclerosis.     

Quantitative proteomic analysis of mitochondrial proteins reveals prosurvival mechanisms in the perpetuation of radiation-induced genomic instability

Radiation-induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear; however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation-induced genomic instability we have evaluated the mitochondrial subproteome and performed quantitative mass spectrometry analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and upregulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype, and evaluation of gene and microRNA expression suggests that epigenetics play a role in the phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under suboptimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

Klotho protects the heart from hyperglycemia-induced injury by inactivating ROS and NF-κB-mediated inflammation both in vitro and in vivo

Cardiac inflammation and oxidative stress play a key role in the pathogenesis of diabetic cardiomyopathy (DCM). The anti-aging protein Klotho has been found to protect cells from inflammation and oxidative stress. The current study aimed to explore the cardioprotective effects of Klotho on DCM and the underlying mechanisms. H9c2 cells and neonatal cardiomyocytes were incubated with 33 mM glucose in the presence or absence of Klotho. Klotho pretreatment effectively inhibited high glucose-induced inflammation, ROS generation, apoptosis, mitochondrial dysfunction, fibrosis and hypertrophy in both H9c2 cells and neonatal cardiomyocytes. In STZ-induced type 1 diabetic mice, intraperitoneal injection of Klotho at 0.01 mg/kg per 48 h for 3 months completely suppressed cardiac inflammatory cytokines and oxidative stress and prevented cardiac cell death and remodeling, which subsequently improved cardiac dysfunction without affecting hyperglycemia. This study revealed that Klotho may exert its protective effects by augmenting nuclear factor erythroid 2-related factor 2 (Nrf2) expression and inactivating nuclear factor κB (NF-κB) activation both in vitro and in vivo. Thus, this work demonstrated for the first time that the anti-aging protein Klotho may be a potential therapeutic agent to treat DCM by inhibiting oxidative stress and inflammation. We also demonstrated the critical roles of the Nrf2 and NF-κB pathways in diabetes-stimulated cardiac injuries and indicated that they may be key therapeutic targets for diabetic complications.     

Mitochondrial Dysfunction Promotes Breast Cancer Cell Migration and Invasion through HIF1α Accumulation via Increased Production of Reactive Oxygen Species

Although mitochondrial dysfunction has been observed in various types of human cancer cells, the molecular mechanism underlying mitochondrial dysfunction mediated tumorigenesis remains largely elusive. To further explore the function of mitochondria and their involvement in the pathogenic mechanisms of cancer development, mitochondrial dysfunction clones of breast cancer cells were generated by rotenone treatment, a specific inhibitor of mitochondrial electron transport complex I. These clones were verified by mitochondrial respiratory defect measurement. Moreover, those clones exhibited increased reactive oxygen species (ROS), and showed higher migration and invasive behaviors compared with their parental cells. Furthermore, antioxidant N-acetyl cysteine, PEG-catalase, and mito-TEMPO effectively inhibited cell migration and invasion in these clones. Notably, ROS regulated malignant cellular behavior was in part mediated through upregulation of hypoxia-inducible factor-1 α and vascular endothelial growth factor. Our results suggest that mitochondrial dysfunction promotes cancer cell motility partly through HIF1α accumulation mediated via increased production of reactive oxygen species.     

The SGLT2 inhibitor Empagliflozin attenuates interleukin-17A-induced human aortic smooth muscle cell proliferation and migration by targeting TRAF3IP2/ROS/NLRP3/Caspase-1-dependent IL-1β and IL-18 secretion

Chronic inflammation and persistent oxidative stress contribute to the development and progression of vascular proliferative diseases. We hypothesized that the proinflammatory cytokine interleukin (IL)-17A induces oxidative stress and amplifies inflammatory signaling in human aortic smooth muscle cells (SMC) via TRAF3IP2-mediated NLRP3/caspase-1-dependent mitogenic and migratory proinflammatory cytokines IL-1β and IL-18. Further, we hypothesized that these maladaptive changes are prevented by empagliflozin (EMPA), an SGLT2 (Sodium/Glucose Cotransporter 2) inhibitor. Supporting our hypotheses, exposure of cultured SMC to IL-17A promoted proliferation and migration via TRAF3IP2, TRAF3IP2-dependent superoxide and hydrogen peroxide production, NLRP3 expression, caspase-1 activation, and IL-1β and IL-18 secretion. Furthermore, NLRP3 knockdown, caspase-1 inhibition, and pretreatment with IL-1β and IL-18 neutralizing antibodies and IL-18BP, each attenuated IL-17A-induced SMC migration and proliferation. Importantly, SMC express SGLT2, and pre-treatment with EMPA attenuated IL-17A/TRAF3IP2-dependent oxidative stress, NLRP3 expression, caspase-1 activation, IL-1β and IL-18 secretion, and SMC proliferation and migration. Importantly, silencing SGLT2 attenuated EMPA-mediated inhibition of IL-17A-induced cytokine secretion and SMC proliferation and migration. EMPA exerted these beneficial antioxidant, anti-inflammatory, anti-mitogenic and anti-migratory effects under normal glucose conditions and without inducing cell death. These results suggest the therapeutic potential of EMPA in vascular proliferative diseases.     

Nitrosonifedipine Ameliorates the Progression of Type 2 Diabetic Nephropathy by Exerting Antioxidative Effects

Diabetic nephropathy (DN) is the major cause of end-stage renal failure. Oxidative stress is implicated in the pathogenesis of DN. Nitrosonifedipine (NO-NIF) is a weak calcium channel blocker that is converted from nifedipine under light exposure. Recently, we reported that NO-NIF has potential as a novel antioxidant with radical scavenging abilities and has the capacity to treat vascular dysfunction by exerting an endothelial protective effect. In the present study, we extended these findings by evaluating the efficacy of NO-NIF against DN and by clarifying the mechanisms of its antioxidative effect. In a model of type 2 DN (established in KKAy mice), NO-NIF administration reduced albuminuria and proteinuria as well as glomerular expansion without affecting glucose metabolism or systolic blood pressure. NO-NIF also suppressed renal and systemic oxidative stress and decreased the expression of intercellular adhesion molecule (ICAM)-1, a marker of endothelial cell injury, in the glomeruli of the KKAy mice. Similarly, NO-NIF reduced albuminuria, oxidative stress, and ICAM-1 expression in endothelial nitric oxide synthase (eNOS) knockout mice. Moreover, NO-NIF suppressed urinary angiotensinogen (AGT) excretion and intrarenal AGT protein expression in proximal tubular cells in the KKAy mice. On the other hand, hyperglycemia-induced mitochondrial superoxide production was not attenuated by NO-NIF in cultured endothelial cells. These findings suggest that NO-NIF prevents the progression of type 2 DN associated with endothelial dysfunction through selective antioxidative effects.     

Ablation of ALCAT1 Mitigates Hypertrophic Cardiomyopathy through Effects on Oxidative Stress and Mitophagy

Oxidative stress causes mitochondrial dysfunction and heart failure through unknown mechanisms. Cardiolipin (CL), a mitochondrial membrane phospholipid required for oxidative phosphorylation, plays a pivotal role in cardiac function. The onset of age-related heart diseases is characterized by aberrant CL acyl composition that is highly sensitive to oxidative damage, leading to CL peroxidation and mitochondrial dysfunction. Here we report a key role of ALCAT1, a lysocardiolipin acyltransferase that catalyzes the synthesis of CL with a high peroxidation index, in mitochondrial dysfunction associated with hypertrophic cardiomyopathy. We show that ALCAT1 expression was potently upregulated by the onset of hyperthyroid cardiomyopathy, leading to oxidative stress and mitochondrial dysfunction. Accordingly, overexpression of ALCAT1 in H9c2 cardiac cells caused severe oxidative stress, lipid peroxidation, and mitochondrial DNA (mtDNA) depletion. Conversely, ablation of ALCAT1 prevented the onset of T4-induced cardiomyopathy and cardiac dysfunction. ALCAT1 deficiency also mitigated oxidative stress, insulin resistance, and mitochondrial dysfunction by improving mitochondrial quality control through upregulation of PINK1, a mitochondrial GTPase required for mitochondrial autophagy. Together, these findings implicate a key role of ALCAT1 as the missing link between oxidative stress and mitochondrial dysfunction in the etiology of age-related heart diseases.     

Mdivi-1 Protects Epileptic Hippocampal Neurons from Apoptosis via Inhibiting Oxidative Stress and Endoplasmic Reticulum Stress in Vitro

Mitochondrial division inhibitor 1 (mdivi-1), a selective inhibitor of the mitochondrial fission protein dynamin-related protein 1, has been proposed to have a neuroprotective effect on hippocampal neurons in animal models of epilepsy. However, the effect of mdivi-1 on epileptic neuronal death in vitro remains unknown. Therefore, we investigated the effect of mdivi-1 and the underlying mechanisms in the hippocampal neuronal culture (HNC) model of acquired epilepsy (AE) in vitro. We found that mitochondrial fission was increased in the HNC model of AE and inhibition of mitochondrial fission by mdivi-1 significantly decreased neuronal apoptosis induced by AE. In addition, mdivi-1 pretreatment significantly attenuated oxidative stress induced by AE characterized by decrease of reactive oxygen species (ROS) production and malondialdehyde level and by increase of superoxide dismutase activity. Moreover, mdivi-1 pretreatment significantly decreased endoplasmic reticulum (ER) stress markers glucose-regulated protein 78, C/EBP homologous protein expression and caspase-3 activation. Altogether, our findings suggest that mdivi-1 protected against AE-induced hippocampal neuronal apoptosis in vitro via decreasing ROS-mediated oxidative stress and ER stress.     

Insulin resistance: Review of the underlying molecular mechanisms

Most human cells utilize glucose as the primary substrate, cellular uptake requiring insulin. Insulin signaling is therefore critical for these tissues. However, decrease in insulin sensitivity due to the disruption of various molecular pathways causes insulin resistance (IR). IR underpins many metabolic disorders such as type 2 diabetes and metabolic syndrome, impairments in insulin signaling disrupting entry of glucose into the adipocytes, and skeletal muscle cells. Although the exact underlying cause of IR has not been fully elucidated, a number of major mechanisms, including oxidative stress, inflammation, insulin receptor mutations, endoplasmic reticulum stress, and mitochondrial dysfunction have been suggested. In this review, we consider the role these cellular mechanisms play in the development of IR.